Ligands for the HisB10 Zn2 + sites of the R-state insulin hexamer

ABSTRACT

The present invention relates to novel ligands for the His B10  Zn 2+  sites of the R-state insulin hexamer having the formula A-B-C-D-X (III), wherein: A is a chemical group which reversibly binds to a His B10  Zn 2+  sites of an insulin hexamer; B is a linker; C is a fragment consisting of 0 to 5 neutral amino acids; D is a fragment comprising 1 to 20 positively charged groups independently selected from amino or guanidino groups; and X is —OH, —NH 2  or a diamino group. The present invention also relates to R-state insulin hexamers comprising such ligands, and aqueous insulin preparations comprising such R-state insulin hexamers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from application serial no.PCT/DK02/00595 filed on Sep. 13, 2002 under 35 U.S.C. 120 and claimspriority under 35 U.S.C. 119 of Danish application no. PA 2001 01337filed on Sep. 14, 2001, Danish application no. PA 2002 01066 filed onJul. 5, 2002, U.S. application Ser. No. 60/323,925, filed 21 Sep. 2001,U.S. application Ser. No. 60/396,051, filed 10 Jul. 2002, the contentsof which are fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention discloses novel ligands for the His10 Zn²⁺ sitesof the R-state insulin hexamer, R-state insulin hexamers comprising suchligands, and aqueous insulin preparations comprising such R-stateinsulin hexamers. The novel preparations release insulin slowlyfollowing subcutaneous injection.

BACKGROUND OF THE INVENTION

Insulin Allostery. The insulin hexamer is an allosteric protein thatexhibits both positive and negative cooperativity and half-of-the-sitesreactivity in ligand binding. This allosteric behaviour consists of twointerrelated allosteric transitions designated L^(A) ₀ and L^(B) ₀,three interconverting allosteric conformation states (eq. 1),

$\begin{matrix}{{T_{6}\overset{L_{0}^{A}}{\longleftrightarrow}T_{3}}{R_{3}\overset{L_{0}^{B}}{\longleftrightarrow}R_{6}}} & (1)\end{matrix}$designated T₆, T₃R₃, and R₆ and two classes of allosteric ligand bindingsites designated as the phenolic pockets and the His^(B10) anion sites.These allosteric sites are associated only with insulin subunits in theR conformation.

Insulin Hexamer Structures and Ligand Binding. The T- to R-transition ofthe insulin hexamer involves transformation of the first nine residuesof the B chain from an extended conformation in the T-state to anα-helical conformation in the R-state. This coil-to-helix transitioncauses the N-terminal residue, Phe^(B1), to undergo an ˜30 Å change inposition. This conformational change creates hydrophobic pockets (thephenolic pockets) at the sub-unit interfaces (three in T₃R₃, and six inR₆), and the new B-chain helices form 3-helix bundles (one in T₃R₃ andtwo in R₆) with the bundle axis aligned along the hexamer three-foldsymmetry axis. The His^(B10) Zn²⁺ in each R₃ unit is forced to changecoordination geometry from octahedral to either tetrahedral (monodentateligands) or pentahedran (bidentate ligands). Formation of the helixbundle creates a narrow hydrophobic tunnel in each R₃ unit that extendsfrom the surface ˜12 Å down to the His^(B10) metal ion. This tunnel andthe His^(B10) Zn²⁺ ion form the anion binding site.

Hexamer Ligand Binding and Stability of Insulin Formulations. The invivo role of the T to R transition is unknown. However, the addition ofallosteric ligands (e.g. phenol and chloride ion) to insulinpreparations is widely used. Hexamerization is driven by coordination ofZn²⁺ at the His^(B10) sites to give T₆, and the subsequentligand-mediated transition of T₆ to T₃R₃ and to R₆ is known to greatlyenhance the physical and chemical stability of the resultingformulations.

Ligand Binding and Long Acting Insulin Formulations. Although theconversion of T₆ to T₃R₃ and R₆ improves the stability of thepreparation, the rate of absorption following subcutaneous injection ofa soluble hexameric preparation is not much affected by the addition ofphenol and chloride.

Putative events following injection of a soluble hexameric preparation.The small molecule ligands initially diffuse away from the protein. Theaffinity of the ligands for insulin may help to slow this process. Onthe other hand, the affinity of Zn²⁺ for e.g. albumin and the largeeffective space available for diffusion of the lipophilic phenol willtend to speed up the separation. In about 10-15 minutes after injection,the distribution of insulin species in the subcutaneous tissue willroughly correspond to that of a zinc-free insulin preparation at thesame dilution. Then, the equilibrium distribution of species at thispoint will determine the observed absorption rate. In this regimen,absorption rates vary between about 1 hour (for rapid-acting insulinanalogues, such as Asp^(B28) human insulin) and about 4 hours(Co³⁺-hexamer).

Current Approaches Toward Slow Acting Insulins. The inherent limitationof the absorption half-life to about 4 hours for a soluble human insulinhexamer necessitates further modifications to obtain the desiredprotraction. Traditionally, this has been achieved by the use ofpreparations wherein the constituent insulin is in the form of acrystalline and/or amorphous precipitate. In this type of formulation,the dissolution of the precipitate in the subcutaneous depot becomesrate-limiting for the absorption. NPH and Ultralente belong to thiscategory of insulin preparations where crystallization/precipitation iseffected by the addition of protamine and excessive zinc ion,respectively.

Another approach involves the use of insulin derivatives where the netcharge is increased to shift the isoelectric point, and hence the pH ofminimum solubility, from about 5.5 to the physiological range. Suchpreparations may be injected as clear solutions at slightly acidic pH.The subsequent adjustment of the pH to neutral inducescrystallization/precipitation in the subcutaneous depot and dissolutionagain becomes rate-limiting for the absorption.Gly^(A21)Arg^(B31)Arg^(B32) human insulin belongs to this category ofinsulin analogues.

Most recently, a series of soluble insulin derivatives with ahydrophobic moiety covalently attached to the side chain of Lys^(B29)have been synthesized. These derivatives may show prolonged actionprofile due to various mechanisms including albumin binding (e.g.B29-N^(ε)-myristoyl-des(B30) human insulin), extensive proteinself-association and/or stickiness (e.g.B29-N^(ε)-(N-lithocholyl-γ-glutamyl)-des(B30) human insulin) induced bythe attached hydrophobic group.

SUMMARY OF THE INVENTION

The present invention provides novel ligands for the His^(B10) Zn²⁺sites of the R-state insulin hexamer. The ligands stabilize the hexamersand modify solubility in the neutral range. The resulting preparationsrelease insulin slowly following subcutaneous injection. In comparisonwith earlier slow release preparations, the present ligands work tomodify the timing of both human insulin and insulin mutants/analogues.The ligands alone or in combination with new ligands for the phenolcavity also confer increased physical and chemical stability of theresulting preparations. Moreover, the preparations release activeinsulin more reproducibly that e.g. NPH preparations.

DEFINITIONS

The following is a detailed definition of the terms used to describe theinvention:

“Halogen” designates an atom selected from the group consisting of F,Cl, Br and I.

The term “C₁-C₆-alkyl” as used herein represents a saturated, branchedor straight hydrocarbon group having from 1 to 6 carbon atoms.Representative examples include, but are not limited to, methyl, ethyl,n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl,isopentyl, neopentyl, tert-pentyl, n-hexyl, isohexyl and the like.

The term “C₁-C₆-alkylene” as used herein represents a saturated,branched or straight bivalent hydrocarbon group having from 1 to 6carbon atoms. Representative examples include, but are not limited to,methylene, 1,2-ethylene, 1,3-propylene, 1,2-propylene, 1,4-butylene,1,5-pentylene, 1,6-hexylene, and the like.

The term “C₂-C₆-alkenyl” as used herein represents a branched orstraight hydrocarbon group having from 2 to 6 carbon atoms and at leastone double bond. Examples of such groups include, but are not limitedto, vinyl, 1-propenyl, 2-propenyl, iso-propenyl, 1,3-butadienyl,1-butenyl, 2-butenyl, 3-butenyl, 2-methyl-1-propenyl, 1-pentenyl,2-pentenyl, 3-pentenyl, 4-pentynyl, 3-methyl-2-butenyl, 1-hexenyl,2-hexenyl, 3-hexenyl, 2,4-hexadienyl, 5-hexenyl and the like.

The term “C₂-C₆-alkynyl” as used herein represents a branched orstraight hydrocarbon group having from 2 to 6 carbon atoms and at leastone triple bond. Examples of such groups include, but are not limitedto, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl,1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl,3-hexynyl, 4-hexynyl, 5-hexynyl, 2,4-hexadienyl and the like.

The term “C₁-C₆-alkoxy” as used herein refers to the radical—O—C₁-C₆-alkyl, wherein C₁-C₆-alkyl is as defined above. Representativeexamples are methoxy, ethoxy, n-propoxy, isopropoxy, butoxy, sec-butoxy,tert-butoxy, pentoxy, isopentoxy, hexoxy, isohexoxy and the like.

The term “C₃-C₈-cycloalkyl” as used herein represents a saturated,carbocyclic group having from 3 to 8 carbon atoms. Representativeexamples are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl and the like.

The term “C₄₋₈-cycloalkenyl” as used herein represents a non-aromatic,carbocyclic group having from 4 to 8 carbon atoms containing one or twodouble bonds. Representative examples are 1-cyclopentenyl,2-cyclopentenyl, 3-cyclopentenyl, 1-cyclohexenyl, 2-cyclohexenyl,3-cyclohexenyl, 2-cycloheptenyl, 3-cycloheptenyl, 2-cyclooctenyl,1,4-cyclooctadienyl and the like.

The term “heterocyclyl” as used herein represents a non-aromatic 3 to 10membered ring containing one or more heteroatoms selected from nitrogen,oxygen and sulphur and optionally containing one or two double bonds.Representative examples are pyrrolidinyl, piperidyl, piperazinyl,morpholinyl, thiomorpholinyl, aziridinyl, tetrahydrofuranyl and thelike.

The term “aryl” as used herein is intended to include carbocyclic,aromatic ring systems such as 6 membered monocyclic and 9 to 14 memberedbi- and tricyclic, carbocyclic, aromatic ring systems. Representativeexamples are phenyl, biphenylyl, naphthyl, anthracenyl, phenanthrenyl,fluorenyl, indenyl, azulenyl and the like. Aryl is also intended toinclude the partially hydrogenated derivatives of the ring systemsenumerated above. Non-limiting examples of such partially hydrogenatedderivatives are 1,2,3,4-tetrahydronaphthyl, 1,4-dihydronaphthyl and thelike.

The term “arylene” as used herein is intended to include divalent,carbocyclic, aromatic ring systems such as 6 membered monocyclic and 9to 14 membered bi- and tricyclic, divalent, carbocyclic, aromatic ringsystems. Representative examples are phenylene, biphenylylene,naphthylene, anthracenylene, phenanthrenylene, fluorenylene, indenylene,azulenylene and the like. Arylene is also intended to include thepartially hydrogenated derivatives of the ring systems enumerated above.Non-limiting examples of such partially hydrogenated derivatives are1,2,3,4-tetrahydronaphthylene, 1,4-dihydronaphthylene and the like.

The term “aryloxy” as used herein denotes a group —O-aryl, wherein arylis as defined above.

The term “aroyl” as used herein denotes a group —C(O)-aryl, wherein arylis as defined above.

The term “heteroaryl” as used herein is intended to include aromatic,heterocyclic ring systems containing one or more heteroatoms selectedfrom nitrogen, oxygen and sulphur such as 5 to 7 membered monocyclic and8 to 14 membered bi- and tricyclic aromatic, heterocyclic ring systemscontaining one or more heteroatoms selected from nitrogen, oxygen andsulphur. Representative examples are furyl, thienyl, pyrrolyl,pyrazolyl, 3-oxopyrazolyl, oxazolyl, thiazolyl, imidazolyl, isoxazolyl,isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, pyranyl, pyridyl,pyridazinyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl,1,3,5-triazinyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl,1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,3-thiadiazolyl,1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, tetrazolyl,thiadiazinyl, indolyl, isoindolyl, benzofuryl, benzothienyl, indazolyl,benzimidazolyl, benzthiazolyl, benzisothiazolyl, benzoxazolyl,benzisoxazolyl, purinyl, quinazolinyl, quinolizinyl, quinolinyl,isoquinolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl,azepinyl, diazepinyl, acridinyl, thiazolidinyl, 2-thiooxothiazolidinyland the like. Heteroaryl is also intended to include the partiallyhydrogenated derivatives of the ring systems enumerated above.Non-limiting examples of such partially hydrogenated derivatives are2,3-dihydrobenzofuranyl, pyrrolinyl, pyrazolinyl, indolinyl,oxazolidinyl, oxazolinyl, oxazepinyl and the like.

The term “heteroarylene” as used herein is intended to include divalent,aromatic, heterocyclic ring systems containing one or more heteroatomsselected from nitrogen, oxygen and sulphur such as 5 to 7 memberedmonocyclic and 8 to 14 membered bi- and tricyclic aromatic, heterocyclicring systems containing one or more heteroatoms selected from nitrogen,oxygen and sulphur. Representative examples are furylene, thienylene,pyrrolylene, oxazolylene, thiazolylene, imidazolylene, isoxazolylene,isothiazolylene, 1,2,3-triazolylene, 1,2,4-triazolylene, pyranylene,pyridylene, pyridazinylene, pyrimidinylene, pyrazinylene,1,2,3-triazinylene, 1,2,4-triazinylene, 1,3,5-triazinylene,1,2,3-oxadiazolylene, 1,2,4-oxadiazolylene, 1,2,5-oxadiazolylene,1,3,4-oxadiazolylene, 1,2,3-thiadiazolylene, 1,2,4-thiadiazolylene,1,2,5-thiadiazolylene, 1,3,4-thiadiazolylene, tetrazolylene,thiadiazinylene, indolylene, isoindolylene, benzofurylene,benzothienylene, indazolylene, benzimidazolylene, benzthiazolylene,benzisothiazolylene, benzoxazolylene, benzisoxazolylene, purinylene,quinazolinylene, quinolizinylene, quinolinylene, isoquinolinylene,quinoxalinylene, naphthyridinylene, pteridinylene, carbazolylene,azepinylene, diazepinylene, acridinylene and the like. Heteroaryl isalso intended to include the partially hydrogenated derivatives of thering systems enumerated above. Non-limiting examples of such partiallyhydrogenated derivatives are 2,3-dihydrobenzofuranylene, pyrrolinylene,pyrazolinylene, indolinylene, oxazolidinylene, oxazolinylene,oxazepinylene and the like.

“Aryl-C₁-C₆-alkyl”, “heteroaryl-C₁-C₆-alkyl”, “aryl-C₂-C₆-alkenyl” etc.is intended to mean C₁-C₆-alkyl or C₂-C₆-alkenyl as defined above,substituted by an aryl or heteroaryl as defined above, for example:

The term “optionally substituted” as used herein means that the groupsin question are either unsubstituted or substituted with one or more ofthe substituents specified. When the groups in question are substitutedwith more than one substituent the substituents may be the same ordifferent.

Furthermore, when polycyclic structures are substituted with one or moresubstituents, it is intended that substitutions at any availableposition in either of the rings that are part of the polycyclicstructure are included.

Certain of the above defined terms may occur more than once in thestructural formulae, and upon such occurrence each term shall be definedindependently of the other.

Furthermore, when using the terms “independently are” and “independentlyselected from” it should be understood that the groups in question maybe the same or different.

The term “treatment” as used herein means the management and care of apatient for the purpose of combating a disease, disorder or condition.The term is intended to include the delaying of the progression of thedisease, disorder or condition, the alleviation or relief of symptomsand complications, and/or the cure or elimination of the disease,disorder or condition. The patient to be treated is preferably a mammal,in particular a human being.

The term “fragment” as used herein is intended to mean a bivalentchemical group

The term “Neutral amino acid” as used herein is intended to mean anynatural (codable) and non-natural amino acid, including α- orβ-aminocarboxylic acids, including D-isomers of these (when applicable)without charges at physiologically relevant pH in the side chain, suchas glycine, alanine, β-alanine, valine, leucine, isoleucine,phenylalanine, tyrosine, aspargine, glutamine, cysteine, methionine,3-aminobenzoic acid, 4-aminobenzoic acid or the like.

The term “positively charged group” as used herein is intended to meanany pharmaceutically acceptable group that contains a positive charge atphysiologically relevant pH, such as amino (primary, secondary andtertiary), ammonium and guanidino groups.

The term “α amino acid” as used herein is intended to mean mean anynatural (codable) and non-natural α-aminocarboxylic acid, includingD-isomers of these.

The term “β amino acid” as used herein is intended to mean anyβ-aminocarboxylic acid, such as β-alanine, isoserine or the like.

When in the specification or claims mention is made of groups ofcompounds such as carboxylates, dithiocarboxylates, phenolates,thiophenolates, alkylthiolates, sulfonamides, imidazoles, triazoles,4-cyano-1,2,3-triazoles, benzimidazoles, benzotriazoles, purines,thiazolidinediones, tetrazoles, 5-mercaptotetrazoles, rhodanines,N-hydroxyazoles, hydantoines, thiohydantoines, naphthoic acids andsalicylic acids, these groups of compounds are intended to include alsoderivatives of the compounds from which the groups take their name.

The term human insulin as used herein refers to naturally producedinsulin or recombinantly produced insulin. Recombinant human insulin maybe produced in any suitable host cell, for example the host cells may bebacterial, fungal (including yeast), insect, animal or plant cells. Theexpression “insulin derivative” as used herein (and related expressions)refers to human insulin or an analogue thereof in which at least oneorganic substituent is bound to one or more of the amino acids.

By “analogue of human insulin” as used herein (and related expressions)is meant human insulin in which one or more amino acids have beendeleted and/or replaced by other amino acids, including non-codeableamino acids, or human insulin comprising additional amino acids, i.e.more than 51 amino acids, such that the resulting analogue possessesinsulin activity.

The term “phenolic compound” or similar expressions as used hereinrefers to a chemical compound in which a hydroxyl group is bounddirectly to a benzene or substituted benzene ring. Examples of suchcompounds include, but are not limited to, phenol, o-cresol, m-cresoland p-cresol.

The term “physiologically relevant pH” as used herein is intended tomean a pH of about 7.1 to 7.9.

When calculating the ratio between precipitated and dissolved insulin indual-acting insulin composition, i.e. a composition containing bothrapid-acting insulin and insulin with a prolonged action, the term“precipitated insulin” as used herein is intended to mean insulinmonomer which is part of a hexamer to which a ligand of the presentinvention is bound at physiologically relevant pH as defined above.Similarly the term “dissolved insulin” as used herein is intended tomean insulin which is not precipitated as defined above.

Abbreviations: 4H3N 4-hydroxy-3-nitrobenzoic acid Abz Aminobenzoic acidAcOH acetic acid BT Benzotriazol-5-oyl DMF N,N-Dimethylformamide DMSODimethylsulfoxide DIC Diisopropylcarbodiimide EDAC1-ethyl-3-(3′-dimethylamino-propyl)carbodiimide, hydrochloride Fmoc9H-Fluorene-9-ylmethoxycarbonyl G, Gly Glycine HOAt1-hydroxy-7-azabenzotriazole HOBT 1-Hydroxybenzotriazole K, Lys LysineNMP N-methyl-2-pyrrolidone Pbf2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl Pmc2,2,5,7,8-pentamethylchroman-6-sulfonyl R, Arg Arginine TFATrifluoroacetic acid

Abbreviations for non-natural amino acid residues:

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: Effect of BTG₂R₅—NH₂ (SEQ ID NO: 1) on pH-solubility profile ofan insulin preparation.

FIG. 2: Effect of BTG₂R₄—NH₂ (SEQ ID NO: 2) on the pH-solubility profileof an insulin preparation.

FIG. 3: Disappearance from the subcutaneous depot (pig model) of insulinpreparations in the presence of BT-AbzG₂R₅—NH₂ (SEQ ID NO: 3) withphenol and 7-hydroxy indole (a-b); and BT-G₂R₅—NH₂ (SEQ ID NO: 1) andBT-G₂R₄ (SEQ ID NO: 2) with phenol (c-d). The bottom panels (e-f) showslow- and dual release profiles, respectively, obtained from Asp^(B28)human insulin formulated with variable concentration of TZD-Abz-G₂R₅(SEQ ID NO: 4)

DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that the two knownligand binding sites of the R-state insulin hexamer can be used toobtain an insulin preparation having prolonged action designed forflexible injection regimes including once-daily, based on insulinmolecules of any kind, e.g. human Insulin or AspB28 human insulin.

The basic concept underlying the present invention involves reversibleattachment of a ligand to the His^(B10) Zn²⁺ site of the R-statehexamer. A suitable ligand binds to the hexamer metal site with one endwhile other moieties are covalently attachment to the other end. On thisbasis, prolonged action via modification of preparation solubility maybe obtained in a number of ways. However, all cases involve the samepoint of protein-ligand attachment and the delivery of human insulin (oranalogues or derivatives thereof) as the active species.

The anions currently used in insulin formulations as allosteric ligandsfor the R-state hexamers (notably chloride ion) bind only weakly to theHis^(B10) anion site. The present invention, which is based on thediscovery of suitable higher affinity ligands for these anion sites,provides ligands which are extended to modify timing via changes inhexamer solubility as outlined above.

Most ligand binding sites in proteins are highly asymmetric. Because theHis^(B10) Zn²⁺ sites reside on the three-fold symmetry axis, these sitesposses a symmetry that is unusual, but not unique. Several otherproteins have highly symmetric ligand binding sites.

The His^(B10) Zn²⁺ site consists of a tunnel or cavity with atriangular-shaped cross-section that extends ˜12 Å from the surface ofthe hexamer down to the His^(B10) Zn²⁺ ion. The diameter of the tunnelvaries along its length and, depending on the nature of the ligandoccupying the site, the opening can be capped over by the Asn^(B3) andPhe^(B1) side chains. The walls of the tunnel are made up of the sidechains of the amino acid residues along one face each of the threeα-helices. The side chains from each helix that make up the lining ofthe tunnel are Phe^(B1), Asn^(B3), and Leu^(B6). Therefore, except forthe zinc ion, which is coordinated to three His^(B10) residues and ispositioned at the bottom of the tunnel, the site is principallyhydrophobic. Depending on the ligand structure, it may be possible forsubstituents on the ligand to make H-bonding interactions with Asn^(B3)and with the peptide linkage to Cys^(B7).

The present invention originates from a search for compounds withsuitable binding properties by using novel UV-visible and fluorescencebased competition assays described herein which are based on thedisplacement of chromophoric ligands from the R-state His^(B10)-Zn²⁺site by the incoming ligand in question. These compounds will bereferred to as “starter compounds” in the following. These assays areeasily transformed into a high-throughput format capable of handlinglibraries from the initial search of compound databases.

These starter compounds provide the starting point for the task ofconstructing a chemical handle that allows for attachment of thepositively charged fragment D (see below).

Thus, from the structure-activity relationship (SAR) informationobtained from the binding assay(s) it will be apparent for those skilledin the art to modify the starter compounds in question by introductionof a chemical group that will allow for coupling to a peptide containinge.g. one or more arginine or lysine residues. These chemical groupsinclude carboxylic acid (amide bond formation with the peptide),carbaldehyde (reductive alkylation of the peptide), sulfonyl chloride(sulphonamide formation with the peptide) or the like.

The decision where and how to introduce this chemical group can be madein various ways. For example: From the SAR of a series of closelyrelated starter compounds, a suitable position in the starter compoundcan be identified and the chemical group can be attached to thisposition, optionally using a spacer group, using synthesis proceduresknown to those skilled in the art.

Alternatively, this chemical group can be attached (optionally using aspacer group using and synthesis procedures known to those skilled inthe art) to a position on the starter compound remote from theZn²⁺-binding functionality

The zinc-binding ligands of the present invention are characterised bythe following formula (I):A-B-C-D-X  (I)wherein:

-   -   A is a functionality capable of reversibly coordinating to a        His^(B10) Zn²⁺ site of an insulin hexamer;    -   B is a valence bond or a non-naturally occurring amino acid        residue containing an aromatic ring;    -   C is a valence bond or a fragment consisting of 1 to 5 neutral        α- or β-amino acids;    -   D is a fragment containing 1 to 20 positively charged groups        independently selected from amino or guanidino groups,        preferably a fragment consisting of 1 to 20 basic amino acids        independently selected from the group consisting of Lys and Arg        and D-isomers of these; and    -   X is OH, NH₂ or a diamino group.

The length of the zinc-binding ligand should be such that it extendsfrom the His^(B10) Zn²⁺ site to beyond the hexamer surface.

A is preferably a chemical structure selected from the group consistingof carboxylates, dithiocarboxylates, phenolates, thiophenolates,alkylthiolates, sulfonamides, imidazoles, triazoles, benzimidazoles,benzotriazoles, purines, thiazolidinediones, naphthoic acids andsalicylic acids.

More preferably, A comprises a benzotriazole, a 3-hydroxy 2-napthoicacid, a salicylic acid, a tetrazole or a thiazolidinedione structure.

A is advantageously selected from one of the following chemicalstructures:

wherein

-   -   R¹ is hydrogen, fluoro, chloro, bromo or iodo,    -   m is 0 or 1.    -   B is preferably a valence bond or one of the following amino        acid residues:

-   -   C is preferably a valence bond or a fragment consisting of 1 to        5 amino acids independently selected from the group consisting        of neutral amino acids, more preferably from the group of amino        acids consisting of Gly, Ala, Thr, and Ser.

In a particular preferred embodiment, C consists of 1-5 Gly residues or1-5 Ala residues.

-   -   D preferably consists of 1-10 Arg residues.    -   X is preferably OH, NH₂ or

The most preferred specific zinc-binding ligands of the presentinvention are:

-   Benzotriazol-5-ylcarbonyl-Gly-Gly-Arg-Arg-Arg-Arg-Arg-Arg-NH₂ (SEQ    ID NO: 5)-   Benzotriazol-5-ylcarbonyl-Gly-Gly-Arg-Arg-Arg-Arg-Arg-NH₂ (SEQ ID    NO: 1)-   Benzotriazol-5-ylcarbonyl-Gly-Gly-Arg-Arg-Arg-Arg-NH₂ (SEQ ID NO: 2)-   Benzotriazol-5-ylcarbonyl-Gly-Gly-Arg-Arg-Arg-NH₂ (SEQ ID NO: 6)-   Benzotriazol-5-ylcarbonyl-Gly-Arg-Arg-Arg-Arg-Arg-NH₂ (SEQ ID NO: 7)-   Benzotriazol-5-ylcarbonyl-Gly-Gly-Gly-Arg-Arg-Arg-Arg-Arg-NH₂ (SEQ    ID NO: 8)-   Benzotriazol-5-ylcarbonyl-4-Abz-Gly-Gly-Arg-Arg-Arg-Arg-Arg-Arg-NH₂    (SEQ ID NO: 9)-   Benzotriazol-5-ylcarbonyl-4-Abz-Gly-Gly-Arg-Arg-Arg-Arg-Arg-NH₂ (SEQ    ID NO: 10)-   Benzotriazol-5-ylcarbonyl-4-Abz-Gly-Gly-Arg-Arg-Arg-Arg-NH₂ (SEQ ID    NO: 11)-   Benzotriazol-5-ylcarbonyl-4-Abz-Gly-Gly-Arg-Arg-Arg-NH₂ (SEQ ID NO:    12)-   Benzotriazol-5-ylcarbonyl-4-Abz-Arg-Arg-Arg-Arg-Arg-NH₂ (SEQ ID NO:    13)-   Benzotriazol-5-ylcarbonyl-4-Apac-Gly-Gly-Arg-Arg-Arg-Arg-Arg-NH₂    (SEQ ID NO: 14)-   Benzotriazol-5-ylcarbonyl-4-Apac-Gly-Gly-Arg-Arg-Arg-Arg-NH₂ (SEQ ID    NO: 15)-   Benzotriazol-5-ylcarbonyl-4-Apac-Gly-Gly-Arg-Arg-Arg-NH₂ (SEQ ID NO:    16)-   Benzotriazol-5-ylcarbonyl-4-Apac-Arg-Arg-Arg-Arg-Arg-NH₂ (SEQ ID NO:    17)-   Benzotriazol-5-ylcarbonyl-4-Apac-Arg-Arg-Arg-Arg-NH₂ (SEQ ID NO: 18)-   Benzotriazol-5-ylcarbonyl-4-Apac-Arg-Arg-Arg-NH₂    [4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-4-Abz-Gly-Gly-Arg-Arg-Arg-Arg-Arg-NH₂    (SEQ ID NO: 19)-   [3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-4-Abz-Gly-Gly-Arg-Arg-Arg-Arg-Arg-NH₂    (SEQ ID NO: 19)-   4-(2H-Tetrazol-5-yl)benzoyl-Abz-Gly-Gly-Arg-Arg-Arg-Arg-Arg-NH₂ (SEQ    ID NO: 19)

In another embodiment the invention provides a zinc-binding ligand ofthe following general formula (II)A-B-C-D-X  (II)wherein:

-   -   A is a chemical group which reversibly binds to a His^(B10) Zn²⁺        site of an insulin hexamer;    -   B is a linker selected from        -   A valence bond        -   A chemical group G^(B) of the formula —B¹—B²—C(O)—,            —B¹—B²—SO₂—, —B¹—B²—CH₂—, or —B¹—B²—NH—; wherein B¹ is a            valence bond, —O—, —S—, or —NR⁶—;        -   B² is a valence bond, C₁-C₁₈-alkylene, C₂-C₁₈-alkenylene,            C₂-C₁₈-alkynylene, arylene, heteroarylene,            —C₁-C₁₈-alkyl-aryl-, —C₂-C₁₈-alkenyl-aryl-,            —C₂-C₁₈-alkynyl-aryl-, —C(═O)—C₁-C₁₈-alkyl-C(═O),            —C(═O)—C₁-C₁₈-alkenyl-C(═O)—,            —C(═O)—C₁-C₁₈-alkyl-O—C₁-C₁₈-alkyl-C(═O)—,            —C(═O)—C₁-C₁₈-alkyl-S—C₁-C₁₈-alkyl-C(═O)—,            —C(═O)—C₁-C₁₈-alkyl-NR⁶—C₁-C₁₈-alkyl-C(═O)—,            —C(═O)-aryl-C(═O)—, —C(═O)-heteroaryl-C(═O)—;        -   wherein the alkylene, alkenylene, and alkynyl enemoieties            are optionally substituted by —CN, —CF₃, —OCF₃, —OR⁶, or            —NR⁶R⁷ and the arylene and heteroarylene moieties are            optionally substituted by halogen, —C(O)OR⁶, —C(O)H, OCOR⁶,            —SO₂, —CN, —CF₃, —OCF₃, —NO₂, —OR⁶, —NR⁶R⁷, C₁-C₁₈-alkyl, or            C₁-C₁₈-alkanoyl;        -   R⁶ and R⁷ are independently H, C₁-C₄-alkyl;    -   C is a fragment consisting of 1 to 5 neutral α- or β-amino acids    -   D is a fragment comprising 1 to 20 positively charged groups        independently selected from amino or guanidine groups; and    -   X is —OH, —NH₂ or a diamino group,        or a salt thereof with a pharmaceutically acceptable acid or        base, or any optical isomer or mixture of optical isomers,        including a racemic mixture, or any tautomeric forms.

In another embodiment A is a chemical structure selected from the groupconsisting of carboxylates, dithiocarboxylates, phenolates,thiophenolates, alkylthiolates, sulfonamides, imidazoles, triazoles,4-cyano-1,2,3-triazoles, benzimidazoles, benzotriazoles, purines,thiazolidinediones, tetrazoles, 5-mercaptotetrazoles, rhodanines,N-hydroxyazoles, hydantoines, thiohydantoines, barbiturates, naphthoicacids and salicylic acids.

In another embodiment A is a chemical structure selected from the groupconsisting of benzotriazoles, 3-hydroxy 2-napthoic acids, salicylicacids, tetrazoles or thiazolidinediones

In another embodiment A is one of the following structures:

wherein

-   -   X is ═O, ═S or ═NH    -   Y is —S—, —O— or —NH—    -   R⁸ and R¹¹ are independently hydrogen or C₁-C₆-alkyl,    -   R⁹ is hydrogen or C₁-C₆-alkyl or aryl, R⁸ and R⁹ may optionally        be combined to form a double bond,    -   R¹⁰ and R¹² are independently hydrogen, aryl, C₁-C₆-alkyl, or        —C(O)NR¹⁶R¹⁷    -   E and G are independently C₁-C₆-alkylene, arylene,        -aryl-C₁-C₆-alkyl, -aryl-C₂-C₆-alkenyl- or heteroarylene,        wherein the alkylene or alkenylene is optionally substituted        with one or more substituents independently selected from        halogen, —CN, —CF₃, —OCF₃, aryl, —COOH and —NH₂, and the arylene        or heteroarylene is optionally substituted with up to three        substituents R¹³, R¹⁴ and R¹⁵.    -   E and R¹⁰ may be connected through one or two valence bonds, G        and R¹² may be connected through one or two valence bonds;    -   R¹³, R¹⁴ and R¹⁵ are independently selected from        -   hydrogen, halogen, —CN, —CH₂CN, —CHF₂, —CF₃, —OCF₃, —OCHF₂,            —OCH₂CF₃, —OCF₂CHF₂, —S(O)₂CF₃, —OS(O)₂CF₃, —SCF₃, —NO₂,            —OR⁶, —NR¹⁶R¹⁷, —SR¹⁶, —NR¹⁶S(O)₂R¹⁷, —S(O)₂NR¹⁶R¹⁷,            —S(O)NR¹⁶R¹⁷, —S(O)R¹⁶, —S(O)₂R¹⁶, —OS(O)₂R¹⁶, —C(O)NR¹⁶R¹⁷,            —OC(O)NR¹⁶R¹⁷, —NR¹⁶C(O)R¹⁷, —CH₂C(O)NR¹⁶R¹⁷,            —OC₁-C₆-alkyl-C(O)NR¹⁶R¹⁷, —CH₂OR¹⁶, —CH₂OC(O)R¹⁶,            —CH₂NR¹⁶R¹⁷, —OC(O)R¹⁶, —OC₁-C₆-alkyl-C(O)OR¹⁶,            —OC₁-C₆-alkyl-OR¹⁶, —SC₁-C₆-alkyl-C(O)OR¹⁶,            —C₂-C₆-alkenyl-C(═O)OR¹⁶, —NR¹⁶—C(═O)—C₁-C₆-alkyl-C(═O)OR¹⁶,            —NR¹⁶—C(═O)—C₁-C₁₆-alkenyl-C(═)OR¹⁶, —C(═O)OR¹⁶, or            —C₂-C₆-alkenyl-C(═O)R¹⁶,        -   C₁-C₆-alkyl, C₂-C₆-alkenyl or C₂-C₆-alkynyl,        -   which may optionally be substituted with one or more            substituents selected from halogen, —CN, —CF₃, —OCF₃, —OR¹⁶,            and —NR¹⁶R¹⁷        -   aryl, aryloxy, aryloxycarbonyl, aroyl, arylsulfanyl,            aryl-C₁-C₆-alkoxy, aryl-C₁-C₆-alkyl, aryl-C₂-C₆-alkenyl,            aroyl-C₂-C₆-alkenyl, aryl-C₂-C₆-alkynyl, heteroaryl,            heteroaryl-C₁-C₆-alkyl, heteroaryl-C₂-C₆-alkenyl or            heteroaryl-C₂-C₆-alkynyl,        -   of which the cyclic moieties optionally may be substituted            with one or more substituents selected from halogen,            —C(O)OR¹⁶, —CH₂C(O)OR¹⁶, —CH₂OR¹⁶, —CN, —CF₃, —OCF₃, —NO₂,            —OR¹⁶, —NR¹⁶R¹⁷ and C₁-C₆-alkyl,    -   R¹⁶ and R¹⁷ independently are hydrogen, OH, C₁-C₆-alkyl,        aryl-C₁-C₆-alkyl or aryl, wherein the alkyl groups may        optionally be substituted with one or more substituents selected        from halogen, —CN, —CF₃, —OCF₃, —OC₁-C₆-alkyl,        —C(O)OC₁-C₆-alkyl, —COOH and —NH₂, and the aryl groups may        optionally be substituted by halogen, —C(O)OC₁-C₆-alkyl, —COOH,        —CN, —CF₃, —OCF₃, —NO₂, —OH, —OC₁-C₆-alkyl, —NH₂, C(═O) or        C₁-C₆-alkyl; R¹⁶ and R¹⁷ when attached to the same nitrogen atom        may form a 3 to 8 membered heterocyclic ring with the said        nitrogen atom, the heterocyclic ring optionally containing one        or two further heteroatoms selected from nitrogen, oxygen and        sulphur, and optionally containing one or two double bonds

In another embodiment X is ═O or ═S

In another embodiment X is ═O

In another embodiment X is ═S

In another embodiment Y is —O— or —S—

In another embodiment Y is —O—

In another embodiment Y is —S—

In another embodiment E is arylene optionally substituted with up tothree substituents R¹³, R¹⁴ and R¹⁵.

In another embodiment E is phenylene or naphtylene optionallysubstituted with up to three substituents R¹³, R¹⁴ and R¹⁵.

In another embodiment E is heteroarylene optionally substituted with upto three substituents R¹³, R¹⁴ and R¹⁵.

In another embodiment E is indolylene optionally substituted with up tothree substituents R¹³, R¹⁴ and R¹⁵.

In another embodiment R⁸ is hydrogen.

In another embodiment R⁹ is hydrogen.

In another embodiment R⁸ and R⁹ are combined to form a double bond.

In another embodiment R¹⁰ is C₁-C₆-alkyl.

In another embodiment R¹⁰ is methyl.

In another embodiment G is phenylene optionally substituted with up tothree substituents R¹³, R¹⁴ and R¹⁵.

In another embodiment R¹¹ is hydrogen.

In another embodiment R¹² is hydrogen.

In another embodiment R¹³, R¹⁴ and R¹⁵ are independently selected from

-   -   hydrogen, halogen, —NO₂, —OR⁶, —NR¹⁶R¹⁷, —SR¹⁶, —NR¹⁶S(O)₂R¹⁷,        —S(O)₂NR¹⁶R¹⁷, —S(O)NR¹⁶R¹⁷, —S(O)R¹⁶, —S(O)₂R¹⁶, —OS(O)₂R¹⁶,        —NR¹⁶C(O)R¹⁷, —CH₂OR¹⁶, —CH₂OC(O)R¹⁶, —CH₂NR¹⁶R¹⁷,        —OC₁-C₆-alkyl-C(O)OR¹⁶, —OC₁-C₆-alkyl-C(O)NR¹⁶R¹⁷,        —OC₁-C₆-alkyl-OR¹⁶, —SC₁-C₆-alkyl-C(O)OR¹⁶,        —C₂-C₆-alkenyl-C(═O)OR¹⁶, or —C₂-C₆-alkenyl-C(═O)R¹⁶,    -   C₁-C₆-alkyl, C₂-C₆-alkenyl or C₂-C₆-alkynyl,    -   which may optionally be substituted with one or more        substituents selected from halogen, —CN, —CF₃, —OCF₃, —OR¹⁶, and        —NR¹⁶R¹⁷    -   aryl, aryloxy, aroyl, arylsulfanyl, aryl-C₁-C₆-alkoxy,        aryl-C₁-C₆-alkyl, aryl-C₂-C₆-alkenyl, aroyl-C₂-C₆-alkenyl,        aryl-C₂-C₆-alkynyl, heteroaryl, heteroaryl-C₁-C₆-alkyl,    -   of which the cyclic moieties optionally may be substituted with        one or more substituents selected from halogen, —C(O)OR¹⁶,        —CH₂C(O)OR¹⁶, —CH₂OR¹⁶, —CN, —CF₃, —OCF₃, —NO₂, —OR¹⁶, —NR¹⁶R¹⁷        and C₁-C₆-alkyl.

In another embodiment R¹³, R¹⁴ and R¹⁵ are independently selected from

-   -   hydrogen, halogen, —NO₂, —OR⁶, —NR¹⁶R¹⁷, —SR¹⁶, —S(O)₂R¹⁶,        —OS(O)₂R¹⁶, —CH₂OC(O)R¹⁶, —OC(O)R¹⁶, —OC₁-C₆-alkyl-C(O)OR¹⁶,        —OC₁-C₆-alkyl-OR¹⁶, —SC₁-C₆-alkyl-C(O)R¹⁶, —C(O)OR¹⁶, or        —C₂-C₆-alkenyl-C(═O)R¹⁶,    -   C₁-C₆-alkyl or C₁-C₆-alkenyl which may optionally be substituted        with one or more substituents selected from halogen, —CN, —CF₃,        —OCF₃, —OR¹⁶, and —NR¹⁶R¹⁷    -   aryl, aryloxy, aroyl, aryl-C₁-C₆-alkoxy, aryl-C₁-C₆-alkyl,        heteroaryl,    -   of which the cyclic moieties optionally may be substituted with        one or more substituents selected from halogen, —C(O)OR¹⁶,        —CH₂C(O)OR¹⁶, —CH₂OR¹⁶, —CN, —CF₃, —OCF₃, —NO₂, —OR¹⁶, —NR¹⁶R¹⁷        and C₁-C₆-alkyl.

In another embodiment R¹³, R¹⁴ and R¹⁵ are independently selected from

-   -   hydrogen, halogen, —NO₂, —OR⁶, —NR¹⁶R¹⁷, —SR¹⁶, —S(O)₂R¹⁶,        —OS(O)₂R¹⁶, —CH₂OC(O)R¹⁶, —OC(O)R¹⁶, —OC₁-C₆-alkyl-C(O)OR¹⁶,        —OC₁-C₆-alkyl-OR¹⁶, —SC₁-C₆-alkyl-C(O)OR¹⁶, —C(O)OR¹⁶, or        —C₂-C₆-alkenyl-C(═O)R¹⁶,    -   C₁-C₆-alkyl or C₁-C₆-alkenyl which may optionally be substituted        with one or more substituents selected from halogen, —CF₃,        —OR¹⁶, and —NR¹⁶R¹⁷    -   aryl, aryloxy, aroyl, aryl-C₁-C₆-alkoxy, aryl-C₁-C₆-alkyl,        heteroaryl,    -   of which the cyclic moieties optionally may be substituted with        one or more substituents selected from halogen, C(O)OR¹⁶, —CN,        —NO₂, —OR¹⁶, —NR¹⁶R¹⁷ and C₁-C₆-alkyl.

In another embodiment R¹³, R¹⁴ and R¹⁵ are independently selected from

-   -   hydrogen, halogen, —OR¹⁶, —OC₁-C₆-alkyl-C(O)OR¹⁶, or —C(O)OR¹⁶,    -   C₁-C₆-alkyl which may optionally be substituted with one or more        substituents selected from halogen, —OR¹⁶, and —NR¹⁶R¹⁷    -   aryl, aryloxy, aryl-C₁-C₆-alkoxy,    -   of which the cyclic moieties optionally may be substituted with        one or more substituents selected from halogen, C(O)OR¹⁶, OR¹⁶,        and C₁-C₆-alkyl.

In another embodiment R¹⁶ and R¹⁷ independently are hydrogen,C₁-C₆-alkyl, or aryl, wherein the alkyl groups may optionally besubstituted with one or more substituents selected from halogen, —CF₃,—OCF₃, —OC₁-C₆-alkyl, —COOH and —NH₂, and the aryl groups may optionallybe substituted by halogen, —COOH, —CN, —CF₃, —OCF₃, —NO₂, —OH,—OC₁-C₆-alkyl, —NH₂, C(═O) or C₁-C₆-alkyl; R¹⁶ and R¹⁷ when attached tothe same nitrogen atom may form a 3 to 8 membered heterocyclic ring withthe said nitrogen atom, the heterocyclic ring optionally containing oneor two further heteroatoms selected from nitrogen, oxygen and sulphur,and optionally containing one or two double bonds

In another embodiment R¹⁶ and R¹⁷ independently are hydrogen,C₁-C₆-alkyl, or aryl, wherein the alkyl groups may optionally besubstituted with one or more substituents selected from halogen, —CF₃,—OC₁-C₆-alkyl, —COOH and —NH₂, and the aryl groups may optionally besubstituted by halogen, —COOH, —CN, —CF₃, —OCF₃, —OH, —NH₂, orC₁-C₆-alkyl.

In another embodiment A is one of the following structures

wherein

-   -   R²⁰ is hydrogen or C₁-C₆-alkyl,    -   R²¹ is hydrogen or C₁-C₆-alkyl,    -   U and V are a valence bond or C₁-C₆-alkylene optionally        substituted with one or more hydroxy, C₁-C₆-alkyl, or aryl        independently,    -   J is C₁-C₆-alkylene, arylene or heteroarylene, wherein the        arylene or heteroarylene is optionally substituted with up to        three substituents R²², R²³ and R²⁴,    -   L is C₁-C₆-alkylene, arylene or heteroarylene, wherein the        arylene or heteroarylene is optionally substituted with up to        three substituents R²⁵, R²⁶ and R²⁷,    -   R¹⁸, R¹⁹, R²², R²³, R²⁴, R²⁵, R²⁶ and R²⁷ are independently        selected from        -   hydrogen, halogen, —CN, —CH₂CN, —CHF₂, —CF₃, —OCF₃, —OCHF₂,            —OCH₂CF₃, —OCF₂CHF₂, —S(O)₂CF₃, —SCF₃, —NO₂, —OR²⁸,            —NR²⁸R²⁹, —SR²⁸, —NR²⁸S(O)₂R²⁹, —S(O)₂NR²⁸R²⁹, —S(O)NR²⁸R²⁹,            —S(O)R²⁸, —S(O)₂R²⁸, —C(O)NR²⁸R²⁹, —OC(O)NR²⁸R²⁹,            —NR²⁸C(O)R²⁹, —NR²⁸C(O)OR²⁹, —CH₂C(O)NR²⁸R²⁹,            —OCH₂C(O)NR²⁸R²⁹, —CH₂OR²⁸, —CH₂NR²⁸R²⁹, —OC(O)R²⁸,            —OC₁-C₆-alkyl-C(O)OR²⁸, —SC₁-C₆-alkyl-C(O)OR²⁸,            —C₂-C₆-alkenyl-C(═O)OR²⁸, —NR²⁸—C(═O)—C₁-C₆-alkyl-C(═O)OR²⁸,            —NR²⁸—C(═O)—C₁-C₆-alkenyl-C(═O)OR²⁸,            —C(═O)NR²⁸—C₁-C₆-alkyl-C(═O)OR²⁸, —C₁-C₆-alkyl-C(═O)OR²⁸, or            —C(O)OR²⁸,        -   C₁-C₆-alkyl, C₂-C₆-alkenyl or C₂-C₆-alkynyl,        -   which may optionally be substituted with one or more            substituents selected from halogen, —CN, —CF₃, —OCF₃, —OR²⁸,            and —NR²⁸R²⁹        -   aryl, aryloxy, aryloxycarbonyl, aroyl, aryl-C₁-C₆-alkoxy,            aryl-C₁-C₆-alkyl, aryl-C₂-C₆-alkenyl, aryl-C₂-C₆-alkynyl,            heteroaryl, heteroaryl-C₁-C₆-alkyl, heteroaryl-C₂-C₆-alkenyl            or heteroaryl-C₂-C₆-alkynyl,        -   of which the cyclic moieties optionally may be substituted            with one or more substituents selected from halogen,            —C(O)OR²⁸, —CN, —CF₃, —OCF₃, —NO₂, —OR²⁸, —NR²⁸R²⁹ and            C₁-C₆-alkyl,    -   R²⁸ and R²⁹ independently are hydrogen, C₁-C₆-alkyl,        aryl-C₁-C₆-alkyl or aryl, or R²⁸ and R²⁹ when attached to the        same nitrogen atom together with the said nitrogen atom may form        a 3 to 8 membered heterocyclic ring optionally containing one or        two further heteroatoms selected from nitrogen, oxygen and        sulphur, and optionally containing one or two double bonds

In another embodiment U is a valence bond

In another embodiment U is C₁-C₆-alkylene optionally substituted withone or more hydroxy, C₁-C₆-alkyl, or aryl

In another embodiment J is arylene or heteroarylene, wherein the aryleneor heteroarylene is optionally substituted with up to three substituentsR²², R²³ and R²⁴

In another embodiment J is arylene optionally substituted with up tothree substituents R²², R²³ and R²⁴

In another embodiment J is phenylene optionally substituted with up tothree substituents R²², R²³ and R²⁴

In another embodiment R²², R²³ and R²⁴ are independently selected from

-   -   hydrogen, halogen, —CHF₂, —CF₃, —OCF₃, —OCHF₂, —OCH₂CF₃,        —OCF₂CHF₂, —SCF₃, —NO₂, —OR²⁸, —NR²⁸R²⁹, —SR²⁸, —C(O)NR²⁸R²⁹,        —OC(O)NR²⁸R²⁹, —NR²⁸C(O)R²⁹, —NR²⁸C(O)OR²⁹, —CH₂C(O)NR²⁸R²⁹,        —OCH₂C(O)NR²⁸R²⁹, —CH₂OR²⁸, —CH₂NR²⁸R²⁹, —OC(O)R²⁸,        —OC₁-C₆-alkyl-C(O)OR²⁸, —SC₁-C₆-alkyl-C(O)OR²⁸,        —C₂-C₆-alkenyl-C(═O)OR²⁸, —NR²⁸—C(═O)—C₁-C₆-alkyl-C(═O)OR²⁸,        —NR²⁸—C(═O)—C₁-C₆-alkenyl-C(═O)OR²⁸—,        —C(═O)NR²⁸—C₁-C₆-alkyl-C(═O)OR²⁸, C₁-C₆-alkyl-C(═O)OR²⁸, or        —C(O)OR²⁸,    -   C₁-C₆-alkyl, C₂-C₆-alkenyl or C₂-C₆-alkynyl,    -   which may optionally be substituted with one or more        substituents selected from halogen, —CN, —CF₃, —OCF₃, —OR²⁸, and        —NR²⁸R²⁹    -   aryl, aryloxy, aryloxycarbonyl, aroyl, aryl-C₁-C₆-alkoxy,        aryl-C₁-C₆-alkyl, aryl-C₂-C₆-alkenyl, aryl-C₂-C₆-alkynyl,        heteroaryl, heteroaryl-C₁-C₆-alkyl, heteroaryl-C₂-C₆-alkenyl or        heteroaryl-C₂-C₆-alkynyl,    -   of which the cyclic moieties optionally may be substituted with        one or more substituents selected from halogen, —C(O)OR²⁸, —CN,        —CF₃, —OCF₃, —NO₂, —OR²⁸, —NR²⁸R²⁹ and C₁-C₆-alkyl

In another embodiment R²², R²³ and R²⁴ are independently selected from

-   -   hydrogen, halogen, —OCF₃, —OR²⁸, —NR²⁸R²⁹, —SR²⁸, —NR²⁸C(O)R²⁹,        —NR²⁸C(O)OR²⁹, —OC(O)R²⁸, —OC₁-C₆-alkyl-C(O)OR²⁸,        —SC₁-C₆-alkyl-C(O)OR²⁸, —C₂-C₆-alkenyl-C(═O)OR²⁸,        —C(═O)NR²⁸—C₁-C₆-alkyl-C(═O)OR²⁸, —C₁-C₆-alkyl-C(═O)OR²⁸, or        —C(O)OR²⁸,    -   C₁-C₆-alkyl optionally substituted with one or more substituents        selected from halogen, —CN, —CF₃, —OCF₃, —OR²⁸, and —NR²⁸R²⁹    -   aryl, aryloxy, aroyl, aryl-C₁-C₆-alkoxy, aryl-C₁-C₆-alkyl,        heteroaryl, heteroaryl-C₁-C₆-alkyl,    -   of which the cyclic moieties optionally may be substituted with        one or more substituents selected from halogen, —C(O)OR²⁸, —CN,        —CF₃, —OCF₃, —NO₂, —OR²⁸, —NR²⁸R²⁹ and C₁-C₆-alkyl

In another embodiment R²², R²³ and R²⁴ are independently selected from

-   -   hydrogen, halogen, —OCF₃, —OR²⁸, —NR²⁸R²⁹, —SR²⁸, —NR²⁸C(O)R²⁹,        —NR²⁸C(O)OR²⁹, —OC(O)R²⁸, —OC₁-C₆-alkyl-C(O)OR²⁸,        —SC₁-C₆-alkyl-C(O)OR²⁸, —C₂-C₆-alkenyl-C(═O)OR²⁸,        —C(═O)NR²⁸—C₁-C₆-alkyl-C(═O)OR²⁸, —C₁-C₆-alkyl-C(═O)OR²⁸, or        —C(O)OR²⁸,    -   C₁-C₆-alkyl optionally substituted with one or more substituents        selected from halogen, —CN, or —CF₃    -   aryl, aryloxy, aroyl, aryl-C₁-C₆-alkoxy, aryl-C₁-C₆-alkyl,        heteroaryl, heteroaryl-C₁-C₆-alkyl,    -   of which the cyclic moieties optionally may be substituted with        one or more substituents selected from halogen, —C(O)OH, —CN,        —CF₃, —NO₂, or C₁-C₆-alkyl

In another embodiment R²⁰ is hydrogen or methyl

In another embodiment R²⁰ is hydrogen

In another embodiment R²⁸ is hydrogen, C₁-C₆-alkyl or aryl

In another embodiment R²⁸ is hydrogen or C₁-C₆-alkyl

In another embodiment R²⁹ is hydrogen or C₁-C₆-alkyl

In another embodiment V is a valence bond

In another embodiment V is C₁-C₆-alkylene optionally substituted withone or more hydroxy, C₁-C₆-alkyl, or aryl

In another embodiment L is C₁-C₆-alkylene or arylene, wherein thearylene is optionally substituted with up to three substituents R²⁵, R²⁶and R²⁷

In another embodiment L is C₁-C₆-alkyl

In another embodiment L is phenylene optionally substituted with up tothree substituents R²⁵, R²⁶ and R²⁷

In another embodiment R²⁵, R²⁶ and R²⁷ are independently selected from

-   -   hydrogen, halogen, —CHF₂, —CF₃, —OCF₃, —OCHF₂, —OCH₂CF₃,        —OCF₂CHF₂, —SCF₃, —NO₂, —OR²⁸, —NR²⁸R²⁹, —SR²⁸, —C(O)NR²⁸R²⁹,        —OC(O)NR²⁸R²⁹, —NR²⁸C(O)R²⁹, —NR²⁸C(O)OR²⁹, —CH₂C(O)NR²⁸R²⁹,        —OCH₂C(O)NR²⁸R²⁹, —CH₂OR²⁸, —CH₂NR²⁸R²⁹, —OC(O)R²⁸,        —OC₁-C₆-alkyl-C(O)OR²⁸, —SC₁-C₆-alkyl-C(O)OR²⁸,        —C₂-C₆-alkenyl-C(═O)OR²⁸, —NR²⁸—C(═O)—C₁-C₆-alkyl-C(═O)OR²⁸,        —NR²⁸—C(═O)—C₁-C₆-alkenyl-C(═O)OR²⁸—,        —C(═O)NR²⁸—C₁-C₆-alkyl-C(═O)OR²⁸, —C₁-C₆-alkyl-C(═O)OR²⁸, or        —C(O)OR²⁸,    -   C₁-C₆-alkyl, C₂-C₆-alkenyl or C₂-C₆-alkynyl,    -   which may optionally be substituted with one or more        substituents selected from halogen, —CN, —CF₃, —OCF₃, —OR²⁸, and        —NR²⁸R²⁹    -   aryl, aryloxy, aryloxycarbonyl, aroyl, aryl-C₁-C₆-alkoxy,        aryl-C₁-C₆-alkyl, aryl-C₂-C₆-alkenyl, aryl-C₂-C₆-alkynyl,        heteroaryl, heteroaryl-C₁-C₆-alkyl, heteroaryl-C₂-C₆-alkenyl or        heteroaryl-C₂-C₆-alkynyl,    -   of which the cyclic moieties optionally may be substituted with        one or more substituents selected from halogen, —C(O)OR²⁸, —CN,        —CF₃, —OCF₃, —NO₂, —OR²⁸, —NR²⁸R²⁹ and C₁-C₆-alkyl

In another embodiment R²⁵, R²⁶ and R²⁷ are independently selected from

-   -   hydrogen, halogen, —OCF₃, —OR²⁸, —NR²⁸R²⁹, —SR²⁸, —NR²⁸C(O)R²⁹,        —NR²⁸C(O)OR²⁹, —OC(O)R²⁸, —OC₁-C₆-alkyl-C(O)R²⁸,        —SC₁-C₆-alkenyl-C(O)OR²⁸, C₂-C₆-alkenyl-C(═O)OR²⁸,        —C(═O)NR²⁸—C₁-C₆-alkyl-C(═O)OR²⁸, —C₁-C₆-alkyl-C(═O)OR²⁸, or        —C(O)OR²⁸,    -   C₁-C₆-alkyl optionally substituted with one or more substituents        selected from halogen, —CN, —CF₃, —OCF₃, —OR²⁸, and —NR²⁸R²⁹    -   aryl, aryloxy, aroyl, aryl-C₁-C₆-alkoxy, aryl-C₁-C₆-alkyl,        heteroaryl, heteroaryl-C₁-C₆-alkyl,    -   of which the cyclic moieties optionally may be substituted with        one or more substituents selected from halogen, —C(O)OR²⁸, —CN,        —CF₃, —OCF₃, —NO₂, —OR²⁸, —NR²⁸R²⁹ and C₁-C₆-alkyl

In anther embodiment R²⁵, R²⁶ and R²⁷ are independently selected from

-   -   hydrogen, halogen, —OCF₃, —OR²⁸, —NR²⁸R²⁹, —SR²⁸, —NR²⁸C(O)R²⁹,        —NR²⁸C(O)OR²⁹, —OC(O)R²⁸, —OC₁-C₆-alkyl-C(O)OR²⁸,        —SC₁-C₆-alkyl-C(O)OR²⁸, —C₂-C₆-alkenyl-C(═O)OR²⁸,        —C(═O)NR²⁸—C₁-C₆-alkyl-C(═O)OR²⁸, —C₁-C₆-alkyl-C(═O)OR²⁸, or        —C(O)OR²⁸,    -   C₁-C₆-alkyl optionally substituted with one or more substituents        selected from halogen, —CN, or —CF₃    -   aryl, aryloxy, aroyl, aryl-C₁-C₆-alkoxy, aryl-C₁-C₆-alkyl,        heteroaryl, heteroaryl-C₁-C₆-alkyl,    -   of which the cyclic moieties optionally may be substituted with        one or more substituents selected from halogen, —C(O)OH, —CN,        —CF₃, —NO₂, or C₁-C₆-alkyl

In another embodiment R²¹ is hydrogen or methyl

In another embodiment R²¹ is hydrogen

In another embodiment R²⁸ is Hydrogen, C₁-C₆-alkyl or aryl

In another embodiment R²⁸ is Hydrogen or C₁-C₆-alkyl

In another embodiment R²⁹ is Hydrogen or C₁-C₆-alkyl

In another embodiment R¹⁸ and R¹⁹ are independently selected from

-   -   hydrogen, halogen, —CN, —CF₃, —OCF₃, —NO₂, —OR²⁸, —NR²⁸R²⁹,        —SR²⁸, —S(O)R²⁸, —S(O)₂R²⁸, —C(O)NR²⁸R²⁹, —CH₂OR²⁸, —OC(O)R²⁸,        —OC₁-C₆-alkyl-C(O)OR²⁸, —SC₁-C₆-alkyl-C(O)OR²⁸, or —C(O)OR²⁸.    -   C₁-C₆-alkyl, C₂-C₆-alkenyl or C₂-C₆-alkynyl,    -   which may optionally be substituted with one or more        substituents selected from halogen, —CN, —CF₃, —OCF₃, —OR²⁸, and        —NR²⁸R²⁹    -   aryl, aryloxy, aryl-C₁-C₆-alkoxy, aryl-C₁-C₆-alkyl, heteroaryl,        heteroaryl-C₁-C₆-alkyl of which the cyclic moieties optionally        may be substituted with one or more substituents selected from        halogen, —C(O)OR²⁸, —CN, —CF₃, —OCF₃, —NO₂, —OR²⁸, —NR²⁸R²⁹ and        C₁-C₆-alkyl

In another embodiment R¹⁸ and R¹⁹ are independently selected from

-   -   hydrogen, halogen, —CN, —CF₃, —NO₂, —OR²⁸, —NR²⁸R²⁹, or        —C(O)OR²⁸    -   C₁-C₆-alkyl optionally substituted with one or more substituents        selected from halogen, —CN, —CF₃, —OCF₃, —OR²⁸, and —NR²⁸R²⁹    -   aryl, aryloxy, aryl-C₁-C₆-alkyl, heteroaryl, of which the cyclic        moieties optionally may be substituted with one or more        substituents selected from halogen, —C(O)OR²⁸, —CN, —CF₃, —OCF₃,        —NO₂, —OR²⁸, —NR²⁸R²⁹ and C₁-C₆-alkyl

In another embodiment A is a compound of the form M-Q-T-

-   -   wherein M is one of the following structures

-   -   wherein W¹, W², and W³ are independently OH, SH or NH₂ and the        phenyl, naphthalene or benzocarbazole rings are optionally        substituted by one or more R³⁴ independently    -   Q is selected from the following:        -   a valence bond        -   —CH₂N(R³⁰)— or —SO₂N(R³¹)—        -   A compound of the formula

-   -   -    wherein Z¹ is S(O)₂ or CH₂, Z² is N, —O— or —S—, and n is 1            or 2;        -   T is        -   A valence bond        -   C₁-C₆-alkylene, C₂-C₆-alkenylene or C₂-C₆-alkynylene, which            may optionally be substituted with one or more substituents            selected from halogen, —CN, —CF₃, —OCF₃, —OR³², and —NR³²R³³        -   Arylene, -aryloxy-, -aryloxycarbonyl-, -aroyl-,            -aryl-C₁-C₆-alkoxy-, -aryl-C₁-C₆-alkyl-,            -aryl-C₂-C₆-alkenyl-, -aryl-C₂-C₆-alkynyl-, heteroarylene,            -heteroaryl-C₁-C₆-alkyl-, -heteroaryl-C₂-C₆-alkenyl- or            -heteroaryl-C₂-C₆-alkynyl-, wherein the cyclic moieties are            optionally substituted by one or more substituents selected            from halogen, —C(O)OR³², —C(O)H, —CN, —CF₃, —OCF₃, —NO₂,            —OR³²R³³, C₁-C₆-alkyl or C₁-C₆-alkanoyl,

    -   R³² and R³³ independently are hydrogen, C₁-C₆-alkyl,        aryl-C₁-C₆-alkyl or aryl, or R³² and R³³ when attached to the        same nitrogen atom together with the said nitrogen atom may form        a 3 to 8 membered heterocyclic ring optionally containing one or        two further heteroatoms selected from nitrogen, oxygen and        sulphur, and optionally containing one or two double bonds, R³⁰        and R³¹ are independently hydrogen, C₁-C₆-alkyl or        C₁-C₆-alkanoyl.

    -   R³⁴ is hydrogen, halogen, —CN, —CH₂CN, —CHF₂, —CF₃, —OCF₃,        —OCHF₂, —OCH₂CF₃, —OCF₂CHF₂, —S(O)₂CF₃, —SCF₃, —NO₂, —OR³²,        —C(O)R³², —NR³²R³³, —SR³², —NR³²S(O)₂R³³, —S(O)₂NR³²R³³,        —S(O)NR³²R³³, —S(O)R³², —S(O)₂R³², —C(O)NR³²R³³, —OC(O)NR³²R³³,        —NR³²C(O)R³³, —CH₂C(O)NR³²R³³, —OCH₂C(O)NR³²R³³, —CH₂OR³²,        —CH₂NR³²R³³, —OC(O)R³², —OC₁-C₆-alkyl-C(O)OR³²,        —SC₁-C₆-alkyl-C(O)OR³²—C₂-C₆-alkenyl-C(═O)OR³²,        —NR³²—C(═O)—C₁-C₆-alkyl-C(═O)OR³²,        —NR³²—C(═O)—C₁-C₆-alkenyl-C(═O)OR³²—, C₁-C₆-alkyl,        C₁-C₆-alkanoyl or —C(O)OR³²,

In another embodiment M is one of the following structures

In another embodiment M is

In another embodiment M is

In another embodiment the salicylic acid moiety is of the formula

In another embodiment the napthoic acid moiety is of the formula

In another embodiment Q is a valence bond, —CH₂N(R³⁰)—, or —SO₂N(R³¹)—

In another embodiment Q is a valence bond

In another embodiment T is

-   -   A valence bond    -   C₁-C₆-alkylene, C₂-C₆-alkenylene or C₂-C₆-alkynylene,    -   which may optionally be substituted with one or more        substituents selected from halogen, —CN, —CF₃, —OCF₃, —OR³², and        —NR³²R³³    -   Arylene, or heteroarylene, wherein the cyclic moieties are        optionally substituted as defined in claim 70

In another embodiment T is

-   -   A valence bond    -   Arylene, or heteroarylene, wherein the cyclic moieties are        optionally substituted as defined in claim 70

In another embodiment T is phenylene or naphthalene

In another embodiment the cyclic moiety in T is optionally substitutedby halogen, —C(O)OR³², —CN, —CF₃, —OR³², —NR³²R³³, C₁-C₆-alkyl orC₁-C₆-alkanoyl

In another embodiment the cyclic moiety in T is optionally substitutedby halogen, —C(O)OR³², —OR³², —NR³²R³³, C₁-C₆-alkyl or C₁-C₆-alkanoyl

In another embodiment the cyclic moiety in T is optionally substitutedby halogen, —C(O)OR³² or —OR³²

In another embodiment T is a valence bond

In another embodiment R³⁰ and R³¹ are independently hydrogen orC₁-C₆-alkyl

In another embodiment R³⁴ is hydrogen, halogen, —CN, —CF₃, —OCF₃, —SCF₃,—NO₂, —OR³², —C(O)R³², —NR³²R³³, —SR³², —C(O)NR³²R³³, —OC(O)NR³²R³³,—NR³²C(O)R³³, —OC(O)R³², —OC₁-C₆-alkyl-C(O)OR³², —SC₁-C₆-alkyl-C(O)OR³²or —C(O)OR³²

In another embodiment R³⁴ is hydrogen, halogen, —CF₃, —NO₂, —OR³²,—NR³²R³³, —SR³², —NR³²C(O)R³³, or —C(O)OR³²

In another embodiment R³⁴ is hydrogen, halogen, —CF₃, —NO₂, —OR³²,—NR³²R³³, or —NR³²C(O)R³³

In another embodiment R³⁴ is hydrogen, halogen, or —OR³²

In another embodiment R³² and R³³ independently are hydrogen,C₁-C₆-alkyl, or aryl

In another embodiment R³² and R³³ independently are hydrogen orC₁-C₆-alkyl

In another embodiment C consists of 1-5 neutral amino acidsindependently selected from the group consisting of Gly, Ala, Thr, andSer

In another embodiment C consists of 1-5 Gly

In another embodiment G^(B) is of the formula —B¹—B²—C(O)—, —B¹—B²—SO₂—or —B¹—B²—CH₂—, wherein B¹ and B² are as defined above

In another embodiment G^(B) is of the formula —B¹—B²—C(O)—, —B¹—B²—SO₂—or —B¹—B²—NH—, wherein B¹ and B² are as defined above

In another embodiment G^(B) is of the formula —B¹—B²—C(O)—, —B¹—B²—CH₂—or —B¹—B²—NH—, wherein B¹ and B² are as defined above

In another embodiment G^(B) is of the formula —B¹—B²—CH₂—, —B¹—B²—SO₂—or —B¹—B²—NH—, wherein B¹ and B² are as defined above

In another embodiment G^(B) is of the formula —B¹—B²—C(O)— or—B¹—B²—SO₂—, wherein B¹ and B² are as defined above

In another embodiment G^(B) is of the formula —B¹—B²—C(O)— or—B¹—B²—CH₂—, wherein B¹ and B² are as defined above

In another embodiment G^(B) is of the formula —B¹—B²—C(O)— or—B¹—B²—NH—, wherein B¹ and B² are as defined above

In another embodiment G^(B) is of the formula —B¹—B²—CH₂— or—B¹—B²—SO₂—, wherein B¹ and B² are as defined above

In another embodiment G^(B) is of the formula —B¹—B²—NH— or —B¹—B²—SO₂—,wherein B¹ and B² are as defined above

In another embodiment G^(B) is of the formula —B¹—B²—CH₂— or —B¹—B²—NH—,wherein B¹ and B² are as defined above

In another embodiment G^(B) is of the formula —B¹—B²—C(O)—

In another embodiment G^(B) is of the formula —B¹—B²—CH₂—

In another embodiment G^(B) is of the formula —B¹—B²—SO₂—

In another embodiment G^(B) is of the formula —B¹—B²—NH—

In another embodiment B¹ is a valence bond, —O—, or —S—

In another embodiment B¹ is a valence bond, —O—, or —N(R⁶)—

In another embodiment B¹ is a valence bond, —S—, or —N(R⁶)—

In another embodiment B¹ is —O—, —S— or —N(R⁶)—

In another embodiment B¹ is a valence bond or —O—

In another embodiment B¹ is a valence bond or —S—

In another embodiment B¹ is a valence bond or —N(R⁶)—

In another embodiment B¹ is —O— or —S—

In another embodiment B¹ is —O— or —N(R⁶)—

In another embodiment B¹ is —S— or —N(R⁶)—

In another embodiment B¹ is a valence bond

In another embodiment B¹ is —O—

In another embodiment B¹ is —S—

In another embodiment B¹ is —N(R⁶)—

In another embodiment B² is a valence bond, C₁-C₁₈-alkylene,C₂-C₁₈-alkenylene, C₂-C₁₈-alkynylene, arylene, heteroarylene,—C₁-C₁₈-alkyl-aryl-, —C(═O)—C₁-C₁₈-alkyl-C(═O)—,—C(═O)—C₁-C₁₈-alkyl-O—C₁-C₁₈-alkyl-C(═O)—,—C(═O)—C₁-C₁₈-alkyl-S—C₁-C₁₈-alkyl-C(═O)—,—C(═O)—C₁-C₁₈-alkyl-NR⁶—C₁-C₁₈-alkyl-C(═O)—; and the alkylene andarylene moieties are optionally substituted as defined above

In another embodiment B² is a valence bond, C₁-C₁₈-alkylene,C₂-C₁₈-alkenylene, C₂-C₁₈-alkynylene, arylene, heteroarylene,—C₁-C₁₈-alkyl-aryl-, —C(═O)—C₁-C₁₈-alkyl-C(═O)—,—C(═O)—C₁-C₁₈-alkyl-O—C₁-C₁₈-alkyl-C(═O)—, and the alkyl and arylmoieties are optionally substituted as defined above

In another embodiment B² is a valence bond, C₁-C₁₈-alkylene,C₂-C₁₈-alkenylene, C₂-C₁₈-alkynylene, arylene, heteroarylene,—C₁-C₁₈-alkyl-aryl-, —C(═O)—C₁-C₁₈-alkyl-C(═O)—, and the alkylene andarylene moieties are optionally substituted as defined above

In another embodiment B² is a valence bond, C₁-C₁₈-alkylene, arylene,heteroarylene, —C₁-C₁₈-alkyl-aryl-, —C(═O)—C₁-C₁₈-alkyl-C(═O)—, and thealkylene and arylene moieties are optionally substituted as definedabove

In another embodiment B² is a valence bond, C₁-C₁₈-alkylene, arylene,heteroarylene, —C₁-C₁₈-alkyl-aryl-, and the alkylene and arylenemoieties are optionally substituted as defined above

In another embodiment B² is a valence bond, C₁-C₁₈-alkylene, arylene,—C₁-C₁₈-alkyl-aryl-, and the alkylene and arylene moieties areoptionally substituted as defined above

In another embodiment B² is a valence bond or C₁-C₁₈-alkylene, and thealkylene moiety is optionally substituted as defined above

In another embodiment D comprises 1 to 16 positively charged groups

In another embodiment D comprises 1 to 12 positively charged groups

In another embodiment D comprises 1 to 10 positively charged groups

In another embodiment D is a fragment containing basic amino acidsindependently selected from the group consisting of Lys and Arg andD-isomers of these.

In another embodiment the basic amino acid is Arg

In another embodiment X is —OH or —NH₂

In another embodiment X is —NH₂

Also provided by the present invention is an R-state insulin hexamercomprising:

6 molecules of insulin, at least 2 zinc ions, and a zinc-binding ligandaccording to any one of the preceding claims.

In one embodiment the insulin forming the R-state insulin hexamer isselected from the group consisting of human insulin, an analoguethereof, a derivative thereof, and combinations of any of these

In another embodiment the insulin is an analogue of human insulinselected from the group consisting of

-   -   i. An analogue wherein position B28 is Asp, Lys, Leu, Val, or        Ala and position B29 is Lys or Pro; and    -   ii. des(B28-B30), des(B27) or des(B30) human insulin.

In another embodiment the insulin is an analogue of human insulinwherein position B28 is Asp or Lys, and position B29 is Lys or Pro.

In another embodiment the insulin is des(B30) human insulin.

In another embodiment the insulin is a derivative of human insulinhaving one or more lipophilic substituents.

In another embodiment the insulin derivative is selected from the groupconsisting of B29-N^(ε)-myristoyl-des(B30) human insulin,B29-N^(ε)-palmitoyl-des(B30) human insulin, B29-N^(ε)-myristoyl humaninsulin, B29-N^(ε)-palmitoyl human insulin, B28-N^(ε)-myristoylLys^(B28) Pro^(B29) human insulin, B28-N^(ε)-palmitoyl Lys^(B28)Pro^(B29) human insulin, B30-N^(ε)-myristoyl-Thr^(B29)Lys^(B30) humaninsulin, B30-N^(ε)-palmitoyl-Thr^(B29)Lys^(B30) human insulin,B29-N^(ε)-(N-palmitoyl-γ-glutamyl)-des(B30) human insulin,B29-N^(ε)-(N-lithocholyl-γ-glutamyl)-des(B30) human insulin,B29-N^(ε)-(ω-carboxyheptadecanoyl)-des(B30) human insulin andB29-N^(ε)-(ω-carboxyheptadecanoyl) human insulin.

In another embodiment the insulin derivative isB29-N^(ε)-myristoyl-des(B30) human insulin.

In another embodiment the insulin hexamer of the invention furthercomprises at least 3 phenolic molecules.

In another embodiment the invention provides an insulin preparationcomprising R-state insulin hexamers as defined above

In another embodiment the invention provides a method of prolonging theaction of an insulin preparation which comprises adding a zinc-bindingligand as defined above to the insulin preparation.

In another embodiment the invention provides an aqueous insulinpreparation as defined above wherein the ratio between precipitatedinsulin and dissolved insulin is in the range from 99:1 to 1:99.

In another embodiment the ratio between precipitated insulin anddissolved insulin is in the range from 95:5 to 5:95

In another embodiment the ratio between precipitated insulin anddissolved insulin is in the range from 80:20 to 20:80

In another embodiment the ratio between precipitated insulin anddissolved insulin is in the range from 70:30 to 30:70.

In another embodiment the invention provides a zinc-binding ligand ofthe following general formula (III)A-B-C-D-X  (III)wherein:

-   -   A is a chemical group which reversibly binds to a His^(B10) Zn²⁺        site of an insulin hexamer;    -   B is a linker selected from        -   A valence bond        -   A chemical group G^(B) of the formula —B¹—B²—C(O)—,            —B¹—B²—SO₂—, —B¹—B²—CH₂—, or —B¹—B²—NH—; wherein B¹ is a            valence bond, —O—, —S—, or —NR⁶—,        -   B² is a valence bond, C₁-C₁₈-alkylene, C₂-C₁₈-alkenylene,            C₂-C₁₈-alkynylene, arylene, heteroarylene,            —C₁-C₁₈-alkyl-aryl-, —C₂-C₁₈-alkenyl-aryl-,            —C₂-C₁₈-alkynyl-aryl-, —C(═O)—C₁-C₁₈-alkyl-C(═O)—,            —C(═O)—C₁-C₁₈-alkenyl-C(═O)—,            —C(═O)—C₁-C₁₈-alkyl-O—C₁-C₁₈-alkyl-C(═O)—,            —C(═O)—C₁-C₁₈-alkyl-S—C₁-C₁₈-alkyl-C(═O)—,            —C(═O)—C₁-C₁₈-alkyl—NR⁶—C₁-C₁₈-alkyl-C(═O),            —C(═O)aryl-C(═O)—, —C(═O)-heteroaryl-C(═O)—;        -   wherein the alkylene, alkenylene, and alkynylene moieties            are optionally substituted by —CN, —CF₃, —OCF₃, —OR⁶, or            —NR⁶R⁷ and the arylene and heteroarylene moieties are            optionally substituted by halogen, —C(O)OR⁶, —C(O)H, OCOR⁶,            —SO₂, —CN, —CF₃, —OCF₃, —NO₂, —OR⁶, —NR⁶R⁷, C₁-C₁₈-alkyl, or            C₁-C₁₈-alkanoyl;        -   R⁶ and R⁷ are independently H, C₁-C₄-alkyl;    -   C is a fragment consisting of 0 to 5 neutral amino acids,        wherein the individual neutral amino acids are the same or        different    -   D is a fragment comprising 1 to 20 positively charged groups        independently selected from amino or guanidino groups, wherein        the individual positively charged groups are the same or        different; and    -   X is —OH, —NH₂ or a diamino group,        or a salt thereof with a pharmaceutically acceptable acid or        base, or any optical isomer or mixture of optical isomers,        including a racemic mixture, or any tautomeric forms.

In another embodiment of the invention A is a chemical structureselected from the group consisting of carboxylates, dithiocarboxylates,phenolates, thiophenolates, alkylthiolates, sulfonamides, imidazoles,triazoles, 4-cyano-1,2,3-triazoles, benzimidazoles, benzotriazoles,purines, thiazolidinediones, tetrazoles, 5-mercaptotetrazoles,rhodanines, N-hydroxyazoles, hydantoines, thiohydantoines, barbiturates,naphthoic acids and salicylic acids.

In another embodiment of the invention A is a chemical structureselected from the group consisting of benzotriazoles, 3-hydroxy2-napthoic acids, salicylic acids, tetrazoles, thiazolidinediones,5-mercaptotetrazoles, or 4-cyano-1,2,3-triazoles.

In another embodiment of the invention A is

wherein

-   -   X is ═O, ═S or ═NH    -   Y is —S—, —O— or —NH—    -   R⁸ and R¹¹ are independently hydrogen or C₁-C₆-alkyl,    -   R⁹ is hydrogen or C₁-C₆-alkyl or aryl, R⁸ and R⁹ may optionally        be combined to form a double bond,    -   R¹⁰ and R¹² are independently hydrogen, aryl, C₁-C₆-alkyl, or        —C(O)NR¹⁶R¹⁷    -   E and G are independently C₁-C₆-alkylene, arylene,        -aryl-C₁-C₆-alkyl-, -aryl-C₂-C₆-alkenyl- or heteroarylene,        wherein the alkylene or alkenylene is optionally substituted        with one or more substituents independently selected from        halogen, —CN, —CF₃, —OCF₃, aryl, —COOH and —NH₂, and the arylene        or heteroarylene is optionally substituted with up to four        substituents R¹³, R¹⁴, R¹⁵, and R^(15A)    -   E and R¹⁰ may be connected through one or two valence bonds, G        and R¹² may be connected through one or two valence bonds;    -   R¹³, R¹⁴, R¹⁵ and R^(15A) are independently selected from        -   hydrogen, halogen, —CN, —CH₂CN, —CHF₂, —CF₃, —OCF₃, —OCHF₂,            —OCH₂CF₃, —OCF₂CHF₂, —S(O)₂CF₃, —OS(O)₂CF₃, —SCF₃, —NO₂,            —OR¹⁶, —NR¹⁶R¹⁷, —SR¹⁶, —NR¹⁶S(O)₂R¹⁷, —S(O)₂NR¹⁶R¹⁷,            —S(O)NR¹⁶R¹⁷, —S(O)R¹⁶, —S(O)₂R¹⁶, —OS(O)₂R¹⁶, —C(O)NR¹⁶R¹⁷,            —OC(O)NR¹⁶R¹⁷, —NR¹⁶C(O)R¹⁷, —CH₂C(O)NR¹⁶R¹⁷,            —OC₁-C₆-alkyl-C(O)NR¹⁶R¹⁷, —CH₂OR¹⁶, —CH₂OC(O)R¹⁶,            —CH₂NR¹⁶R¹⁷, —OC(O)R¹⁶, —OC₁-C₆-alkyl-C(O)OR¹⁶,            —OC₁-C₆-alkyl-OR¹⁶, —SC₁-C₆-alkyl-C(O)OR¹⁶,            —C₂-C₆-alkenyl-C(═O)OR¹⁶, —NR¹⁶—C(═O)—C₁-C₆-alkyl-C(═O)OR¹⁶,            —NR¹⁶—C(═O)—C₁-C₆-alkenyl-C(═O)OR¹⁶, —C(O)OR¹⁶, or            —C₂-C₆-alkenyl-C(═O)R¹⁶, ═O, or —C₂-C₆-alkenyl-C(═O)NR¹⁶R¹⁷,        -   C₁-C₆-alkyl, C₂-C₆-alkenyl or C₂-C₆-alkynyl,        -   which may optionally be substituted with one or more            substituents selected from halogen, —CN, —CF₃, —OCF₃, —OR¹⁶,            and —NR¹⁶R¹⁷        -   aryl, aryloxy, aryloxycarbonyl, aroyl, arylsulfanyl,            aryl-C₁-C₆-alkoxy, aryl-C₁-C₆-alkyl, aryl-C₂-C₆-alkenyl,            aroyl-C₂-C₆-alkenyl, aryl-C₂-C₆-alkynyl, heteroaryl,            heteroaryl-C₁-C₆-alkyl, heteroaryl-C₂-C₆-alkenyl or            heteroaryl-C₂-C₆-alkynyl,        -   of which the cyclic moieties optionally may be substituted            with one or more substituents selected from halogen,            —C(O)OR¹⁶, —CH₂C(O)O¹⁶, —CH₂OR¹⁶, —CN, —CF₃, —OCF₃, —NO₂,            —OR¹⁶, —NR¹⁶R¹⁷, S(O)₂R¹⁶, aryl and C₁-C₆-alkyl,    -   R¹⁶ and R¹⁷ independently are hydrogen, OH, C₁-C₂₀-alkyl,        aryl-C₁-C₆-alkyl or aryl, wherein the alkyl groups may        optionally be substituted with one or more substituents selected        from halogen, —CN, —CF₃, —OCF₃, —OC₁-C₆-alkyl,        —C(O)OC₁-C₆-alkyl, —COOH and —NH₂, and the aryl groups may        optionally be substituted by halogen, —C(O)OC₁-C₆-alkyl, —COOH,        —CN, —CF₃, —OCF₃, —NO₂, —OH, —OC₁-C₆-alkyl, —NH₂, C(═O) or        C₁-C₆-alkyl; R¹⁶ and R¹⁷ when attached to the same nitrogen atom        may form a 3 to 8 membered heterocyclic ring with the said        nitrogen atom, the heterocyclic ring optionally containing one        or two further heteroatoms selected from nitrogen, oxygen and        sulphur, and optionally containing one or two double bonds

In another embodiment of the invention X is ═O or ═S

In another embodiment of the invention X is ═O

In another embodiment of the invention X is ═S

In another embodiment of the invention Y is —O— or —S—

In another embodiment of the invention Y is —O—

In another embodiment of the invention Y is —S—

In another embodiment of the invention E is arylene optionallysubstituted with up to four substituents, R¹³, R¹⁴, R¹⁵, and R^(15A).

In another embodiment of the invention E is phenylene or naphtyleneoptionally substituted with up to four substituents, R¹³, R¹⁴, R¹⁵, andR^(15A).

In another embodiment of the invention E is

In another embodiment of the invention E is

In another embodiment of the invention E is phenylene

In another embodiment of the invention E is heteroarylene optionallysubstituted with up to four substituents, R¹³, R¹⁴, R¹⁵, and R^(15A).

In another embodiment of the invention E is benzofuranylidene optionallysubstituted with up to four substituents R¹³, R¹⁴, R¹⁵, and R^(15A).

In another embodiment of the invention E is

In another embodiment of the invention E is carbazolylidene optionallysubstituted with up to four substituents R¹³, R¹⁴, R¹⁵, and R^(15A).

In another embodiment of the invention E is

In another embodiment of the invention E is quinolylidene optionallysubstituted with up to four substituents R¹³, R¹⁴, R¹⁵, and R^(15A).

In another embodiment of the invention E is

In another embodiment of the invention E is indolylene optionallysubstituted with up to four substituents R¹³, R¹⁴, R¹⁵, and R^(15A).

In another embodiment of the invention E is

In another embodiment of the invention R⁸ is Hydrogen.

In another embodiment of the invention R⁹ is Hydrogen.

In another embodiment of the invention R⁸ and R⁹ are combined to form adouble bond.

In another embodiment of the invention R¹⁰ is C₁-C₆-alkyl.

In another embodiment of the invention R¹⁰ is methyl.

In another embodiment of the invention G is phenylene optionallysubstituted with up to four substituents, R¹³, R¹⁴, R¹⁵, and R^(15A).

In another embodiment of the invention R¹¹ is Hydrogen.

In another embodiment of the invention R¹² is Hydrogen.

In another embodiment of the invention R¹³, R¹⁴, R¹⁵ and R^(15A) areindependently selected from

-   -   hydrogen, halogen —NO₂, —OR⁶, —NR¹⁶R¹⁷, —SR¹⁶, —NR¹⁶S(O)₂R¹⁷,        —S(O)₂NR¹⁶R¹⁷, —S(O)NR¹⁶R¹⁷, —S(O)R¹⁶, —S(O)₂R¹⁶, —OS(O)₂R¹⁶,        —NR¹⁶C(O)R¹⁷, —CH₂OR¹⁶, —CH₂OC(O)R¹⁶, —CH₂NR¹⁶R¹⁷, —OC(O)R¹⁶,        —OC₁-C₆-alkyl-C(O)OR¹⁶, —OC₁-C₆-alkyl-C(O)NR¹⁶R¹⁷,        —OC₁-C₆-alkyl-OR¹⁶, —SC₁-C₆-alkyl-C(O)OR¹⁶,        —C₂-C₆-alkenyl-C(═O)OR¹⁶, —C(O)OR¹⁶, or —C₂-C₆-alkenyl-C(═O)R¹⁶,    -   C₁-C₆-alkyl, C₂-C₆-alkenyl or C₂-C₆-alkynyl,    -   which may optionally be substituted with one or more        substituents selected from halogen, —CN, —CF₃, —OCF₃, —OR¹⁶, and        —NR¹⁶R¹⁷    -   aryl, aryloxy, aroyl, arylsulfanyl, aryl-C₁-C₆-alkoxy,        aryl-C₁-C₆-alkyl, aryl-C₂-C₆-alkenyl, aroyl-C₂-C₆-alkenyl,        aryl-C₂-C₆-alkynyl, heteroaryl, heteroaryl-C₁-C₆-alkyl,    -   of which the cyclic moieties optionally may be substituted with        one or more substituents selected from halogen, —C(O)OR¹⁶,        —CH₂C(O)OR¹⁶, —CH₂OR¹⁶, —CN, —CF₃, —OCF₃, —NO₂, —OR¹⁶, —NR¹⁶R¹⁷        and C₁-C₆-alkyl.

In another embodiment of the invention R¹³, R¹⁴, R¹⁵ and R^(15A) areindependently selected from

-   -   hydrogen, halogen, —NO₂, —OR⁶, —NR¹⁶R¹⁷, —S¹⁶, —S(O)₂R¹⁶,        —OS(O)₂R¹⁶, —CH₂OC(O)R¹⁶, —OC(O)¹⁶, —OC₁-C₆-alkyl-C(O)OR¹⁶,        —OC₁-C₆-alkyl-OR¹⁶, —SC₁-C₆-alkyl-C(O)OR¹⁶, —C(O)OR¹⁶, or        —C₂-C₆-alkenyl-C(═O)R¹⁶,    -   C₁-C₆-alkyl or C₁-C₆-alkenyl which may optionally be substituted        with one or more substituents selected from halogen, —CN, —CF₃,        —OCF₃, —OR¹⁶, and —NR¹⁶R¹⁷    -   aryl, aryloxy, aroyl, aryl-C₁-C₆-alkoxy, aryl-C₁-C₆-alkyl,        heteroaryl,    -   of which the cyclic moieties optionally may be substituted with        one or more substituents selected from halogen, —C(O)OR¹⁶,        —CH₂C(O)OR¹⁶, —CH₂OR¹⁶, —CN, —CF₃, —OCF₃, —NO₂, —OR¹⁶, —NR¹⁶R¹⁷        and C₁-C₆-alkyl.

In another embodiment of the invention R¹³, R¹⁴, R¹⁵ and R^(15A) areindependently selected from

-   -   hydrogen, halogen, —NO₂, —OR⁶, —NR¹⁶R¹⁷, —SR¹⁶, —S(O)₂R¹⁶,        —OS(O)₂R¹⁶, —CH₂OC(O)R¹⁶, —OC(O)R¹⁶, —OC₁-C₆-alkyl-C(O)OR¹⁶,        —OC₁-C₆-alkyl-OR¹⁶, —SC₁-C₆-alkyl-C(O)OR¹⁶, —C(O)OR¹⁶, or        —C₂-C₆-alkenyl-C(═O)R¹⁸,    -   C₁-C₆-alkyl or C₁-C₆-alkenyl which may optionally be substituted        with one or more substituents selected from halogen, —CF₃,        —OR¹⁶, and —NR¹⁶R¹⁷    -   aryl, aryloxy, aroyl, aryl-C₁-C₆-alkoxy, aryl-C₁-C₆-alkyl,        heteroaryl    -   of which the cyclic moieties optionally may be substituted with        one or more substituents selected from halogen, C(O)OR¹⁶, —CN,        —NO₂, —OR¹⁶, —NR¹⁶R¹⁷ and C₁-C₆-alkyl.

In another embodiment of the invention R¹³, R¹⁴, R¹⁵ and R^(15A) areindependently selected from

-   -   hydrogen, halogen, —OR⁶, —OC₁-C₆-alkyl-C(O)OR¹⁶, or —C(O)OR¹⁶,    -   C₁-C₆-alkyl which may optionally be substituted with one or more        substituents selected from halogen, —OR¹⁶, and —NR¹⁶R¹⁷    -   aryl, aryloxy, aryl-C₁-C₆-alkoxy,    -   of which the cyclic moieties optionally may be substituted with        one or more substituents selected from halogen, C(O)OR¹⁶, OR¹⁶,        and C₁-C₆-alkyl.

In another embodiment of the invention R¹⁶ and R¹⁷ independently arehydrogen, C₁-C₂₀-alkyl, or aryl, wherein the alkyl groups may optionallybe substituted with one or more substituents selected from halogen,—CF₃, —OCF₃, —OC₁-C₆-alkyl, —COOH and —NH₂, and the aryl groups mayoptionally be substituted by halogen, —COOH, —CN, —CF₃, —OCF₃, —NO₂,—OH, —OC₁-C₆-alkyl, —NH₂, C(═O) or C₁-C₆-alkyl; R¹⁶ and R¹⁷ whenattached to the same nitrogen atom may form a 3 to 8 memberedheterocyclic ring with the said nitrogen atom, the heterocyclic ringoptionally containing one or two further heteroatoms selected fromnitrogen, oxygen and sulphur, and optionally containing one or twodouble bonds

In another embodiment of the invention R¹⁶ and R¹⁷ independently arehydrogen, C₁-C₂₀-alkyl, or aryl, wherein the alkyl groups may optionallybe substituted with one or more substituents selected from halogen,—CF₃, —OC₁-C₆-alkyl, —COOH and —NH₂, and the aryl groups may optionallybe substituted by halogen, —COOH, —CN, —CF₃, —OCF₃, —OH, —NH₂, orC₁-C₆-alkyl.

In another embodiment of the invention A is

wherein

-   -   R²⁰ is hydrogen or C₁-C₆-alkyl,    -   R²¹ is hydrogen or C₁-C₆-alkyl,    -   U and V are a valence bond or C₁-C₆-alkylene optionally        substituted with one or more hydroxy, C₁-C₆-alkyl, or aryl        independently,    -   J is C₁-C₆-alkylene, arylene or heteroarylene, wherein the        arylene or heteroarylene is optionally substituted with up to        three substituents R²², R²³ and R²⁴,    -   L is C₁-C₆-alkylene, arylene or heteroarylene, wherein the        arylene or heteroarylene is optionally substituted with up to        three substituents R²⁵, R²⁶ and R²⁷,    -   R¹⁸, R¹⁹, R²², R²³R²⁴, R²⁵, R²⁶ and R²⁷ are independently        selected from        -   hydrogen, halogen, —CN, —CH₂CN, —CHF₂, —CF₃, —OCF₃, —OCHF₂,            —OCH₂CF₃, —OCF₂CHF₂, —S(O)₂CF₃, —SCF₃, —NO₂, —OR²⁸,            —NR²⁸R²⁹, —SR²⁸, —NR²⁸S(O)₂R²⁹, —S(O)₂NR²⁸R²⁹, —S(O)NR²⁸R²⁹,            —S(O)R²⁸, —S(O)₂R²⁸, —C(O)NR²⁸R²⁹, —OC(O)NR²⁸R²⁹,            —NR²⁸C(O)R²⁹, —NR²⁸C(O)R²⁹, —CH₂C(O)NR²⁸R²⁹,            —OCH₂C(O)NR²⁸R²⁹, —CH₂OR²⁸, —CH₂NR²⁸R²⁹, —OC(O)R²⁸,            —OC₁-C₆-alkyl-C(O)OR²⁸, —SC₁-C₆-alkyl-C(O)OR²⁸,            —C₂-C₆-alkenyl-C(═O)OR²⁸, —NR²⁸—C(═O)—C₁-C₆-alkyl-C(═O)OR²⁸,            —NR²⁸—C(═O)—C₁-C₆-alkenyl-C(═O)OR²⁸,            —C(═O)NR²⁸—C₁-C₆-alkyl-C(═O)OR²⁸, —C₁-C₆-alkyl-C(═O)OR²⁸, or            —C(O)OR²⁸,        -   C₁-C₆-alkyl, C₂-C₆-alkenyl or C₂-C₆-alkynyl,        -   which may optionally be substituted with one or more            substituents selected from halogen, —CN, —CF₃, —OCF₃, —OR²⁸,            and —NR²⁸R²⁹        -   aryl, aryloxy, aryloxycarbonyl, aroyl, aryl-C₁-C₆-alkoxy,            aryl-C₁-C₆-alkyl, aryl-C₂-C₆-alkenyl, aryl-C₂-C₆-alkynyl,            heteroaryl, heteroaryl-C₁-C₆-alkyl, heteroaryl-C₂-C₆-alkenyl            or heteroaryl-C₂-C₆-alkynyl,        -   of which the cyclic moieties optionally may be substituted            with one or more substituents selected from halogen,            —C(O)OR²⁸, —CN, —CF₃, —OCF₃, —NO₂, —OR²⁸, —NR²⁸R²⁹ and            C₁-C₆-alkyl,    -   R²⁸ and R²⁹ independently are hydrogen, C₁-C₆-alkyl,        aryl-C₁-C₆-alkyl or aryl, or R²⁸ and R²⁹ when attached to the        same nitrogen atom together with the said nitrogen atom may form        a 3 to 8 membered heterocyclic ring optionally containing one or        two further heteroatoms selected from nitrogen, oxygen and        sulphur, and optionally containing one or two double bonds

In another embodiment of the invention U is a valence bond

In another embodiment of the invention U is C₁-C₆-alkylene optionallysubstituted with one or more hydroxy, C₁-C₆-alkyl, or aryl

In another embodiment of the invention J is arylene or heteroarylene,wherein the arylene or heteroarylene is optionally substituted with upto three substituents R²², R²³ and R²⁴

In another embodiment of the invention J is arylene optionallysubstituted with up to three substituents R²², R²³ and R²⁴

In another embodiment of the invention J is phenylene optionallysubstituted with up to three substituents R²², R²³ and R²⁴

In another embodiment of the invention J is

In another embodiment of the invention R²², R²³ and R²⁴ areindependently selected from

-   -   hydrogen, halogen, —CHF₂, —CF₃, —OCF₃, —OCHF₂, —OCH₂CF₃,        —OCF₂CHF₂, —SCF₃, —NO₂, —OR²⁸, —NR²⁸R²⁹, —SR²⁸—C(O)NR²⁸R²⁹,        —OC(O)NR²⁸R²⁹, —NR²⁸C(O)R²⁹, —NR²⁸C(O)OR²⁹, —CH₂C(O)NR²⁸R²⁹,        —OCH₂C(O)NR²⁸R²⁹, —CH₂OR²⁸, —CH₂NR²⁸R²⁹, —OC(O)R²⁸,        —OC₁-C₆-alkyl-C(O)OR²⁸, —SC₁-C₆-alkyl-C(O)OR²⁸,        —C₂-C₆-alkenyl-C(═O)OR²⁸, —NR²⁸—C(═O)—C₁-C₆-alkyl-C(═O)OR²⁸,        —NR²⁸—C(═O)—C₁-C₆-alkenyl-C(═O)OR²⁸—,        —C(═O)NR²⁸—C₁-C₆-alkyl-C(═O)OR²⁸, —C₁-C₆-alkyl-C(═O)OR²⁸, or        —C(O)OR²⁸,    -   C₁-C₆-alkyl, C₂-C₆-alkenyl or C₂-C₆-alkynyl,    -   which may optionally be substituted with one or more        substituents selected from halogen, —CN, —CF₃, —OCF₃, —OR²⁸, and        —NR²⁸R²⁹    -   aryl, aryloxy, aryloxycarbonyl, aroyl, aryl-C₁-C₆-alkoxy,        aryl-C₁-C₆-alkyl, aryl-C₂-C₆-alkenyl, aryl-C₂-C₆-alkynyl,        heteroaryl, heteroaryl-C₁-C₆-alkyl, heteroaryl-C₂-C₆-alkenyl or        heteroaryl-C₂-C₆-alkynyl,    -   of which the cyclic moieties optionally may be substituted with        one or more substituents selected from halogen, —C(O)OR²⁸, —CN,        —CF₃, —OCF₃, —NO₂, —OR²⁸, —NR²⁸R²⁹ and C₁-C₆-alkyl

In another embodiment of the invention R²², R²³ and R²⁴ areindependently selected from

-   -   hydrogen, halogen, —OCF₃, —OR²⁸, —NR²⁸R²⁹, —SR²⁸, —NR²⁸C(O)R²⁹,        —NR²⁸C(O)OR²⁹, —OC(O)R²⁸, —OC₁-C₆-alkyl-C(O)OR²⁸,        —SC₁-C₆-alkyl-C(O)OR²⁸, —C₂-C₆-alkenyl-C(═O)OR²⁸,        —C(═O)NR²⁸—C₁-C₆-alkyl-C(═O)OR²⁸, —C₁-C₆-alkyl-C(═O)OR²⁸, or        —C(O)OR²⁸,    -   C₁-C₆-alkyl optionally substituted with one or more substituents        selected from halogen, —CN, —CF₃, —OCF₃, —OR²⁸, and —NR²⁸R²⁹    -   aryl, aryloxy, aroyl, aryl-C₁-C₆-alkoxy, aryl-C₁-C₆-alkyl,        heteroaryl, heteroaryl-C₁-C₆-alkyl,    -   of which the cyclic moieties optionally may be substituted with        one or more substituents selected from halogen, —C(O)OR²⁸, —CN,        —CF₃, —OCF₃, —NO₂, —OR²⁸, —NR²⁸R²⁹ and C₁-C₆-alkyl

In another embodiment of the invention R²², R²³ and R²⁴ areindependently selected from

-   -   hydrogen, halogen, —OCF₃, —OR²⁸, —NR²⁸R²⁹, —SR²⁸, —NR²⁸C(O)R²⁹,        —NR²⁸C(O)OR²⁹, —OC(O)R²⁸, —OC₁-C₆-alkyl-C(O)OR²⁸,        —SC₁-C₆-alkyl-C(O)OR²⁸, —C₂-C₆-alkenyl-C(═O)OR²⁸,        —C(═O)NR²⁸—C₁-C₆-alkyl-C(═O)OR²⁸, —C₁-C₆-alkyl-C(═O)OR²⁸, or        —C(O)OR²⁸,    -   C₁-C₆-alkyl optionally substituted with one or more substituents        selected from halogen, —CN, or —CF₃    -   aryl, aryloxy, aroyl, aryl-C₁-C₆-alkoxy, aryl-C₁-C₆-alkyl,        heteroaryl, heteroaryl-C₁-C₆-alkyl,    -   of which the cyclic moieties optionally may be substituted with        one or more substituents selected from halogen, —C(O)OH, —CN,        —CF₃, —NO₂, or C₁-C₆-alkyl

In another embodiment of the invention R²⁰ is hydrogen or methyl

In another embodiment of the invention R²⁰ is hydrogen

In another embodiment of the invention R²⁸ is Hydrogen, C₁-C₆-alkyl oraryl

In another embodiment of the invention R²⁸ is Hydrogen or C₁-C₆-alkyl

In another embodiment of the invention R²⁹ is Hydrogen or C₁-C₆-alkyl

In another embodiment of the invention V is a valence bond

In another embodiment of the invention V is C₁-C₆-alkylene optionallysubstituted with one or more hydroxy, C₁-C₆-alkyl, or aryl

In another embodiment of the invention L is C₁-C₆-alkylene or arylene,wherein the arylene is optionally substituted with up to threesubstituents R²⁵, R²⁶ and R²⁷

In another embodiment of the invention L is C₁-C₆-alkylene

In another embodiment of the invention L is phenylene optionallysubstituted with up to three substituents R²⁵, R²⁶ and R²⁷

In another embodiment of the invention R²⁵, R²⁶ and R²⁷ areindependently selected from

-   -   hydrogen, halogen, —CHF₂, —CF₃, —OCF₃, —OCHF₂, —OCH₂CF₃,        —OCF₂CHF₂, —SCF₃, —NO₂, —OR²⁸, —NR²⁸R²⁹, —SR²⁸, —C(O)NR²⁸R²⁹,        —OC(O)NR²⁸R²⁹, —NR²⁸C(O)R²⁹, —NR²⁸C(O)OR²⁹, —CH₂C(O)NR²⁸R²⁹,        —OCH₂C(O)NR²⁸R²⁹, —CH₂OR²⁸, —CH₂NR²⁸R²⁹, —OC(O)R²⁸,        —OC₁-C₆-alkyl-C(O)OR²⁸, —SC₁-C₆-alkyl-C(O)OR²⁸,        —C₂-C₆-alkenyl-C(═O)OR²⁸, —NR²⁸—C(═O)—C₁-C₆-alkyl-C(═O)OR²⁸,        —NR²⁸—C(═)—C₁-C₆-alkenyl-C(═O)OR²⁸—,        —C(═O)NR²⁸—C₁-C₆-alkyl-C(═O)OR²⁸, —C₁-C₆-alkyl-C(═O)OR²⁸, or        —C(O)OR²⁸,    -   C₁-C₆-alkyl, C₂-C₆-alkenyl or C₂-C₆-alkynyl,    -   which may optionally be substituted with one or more        substituents selected from halogen, —CN, —CF₃, —OCF₃, —OR²⁸, and        —NR²⁸R²⁹    -   aryl, aryloxy, aryloxycarbonyl, aroyl, aryl-C₁-C₆-alkoxy,        aryl-C₁-C₆-alkyl, aryl-C₂-C₆-alkenyl, aryl-C₂-C₆-alkynyl,        heteroaryl, heteroaryl-C₁-C₆-alkyl, heteroaryl-C₂-C₆-alkenyl or        heteroaryl-C₂-C₆-alkynyl,    -   of which the cyclic moieties optionally may be substituted with        one or more substituents selected from halogen, —C(O)OR²⁸, —CN,        —CF₃, —OCF₃, —NO₂, —OR²⁸, —NR²⁸R²⁹ and C₁-C₆-alkyl

In another embodiment of the invention R²⁵, R²⁶ and R²⁷ areindependently selected from

-   -   hydrogen, halogen, —OCF₃, —OR²⁸, —NR²⁸R²⁹, —SR²⁸, —NR²⁸C(O)R²⁹,        —NR²⁸C(O)OR²⁹, —OC(O)R²⁸, —OC₁-C₆-alkyl-C(O)OR²⁸,        —SC₁-C₆-alkyl-C(O)OR²⁸, —C₂-C₆-alkenyl-C(═O)OR²⁸,        —C(═O)NR²⁸—C₁-C₆-alkyl-C(═O)OR²⁸, —C₁-C₆-alkyl-C(═O)OR²⁸, or        —C(O)OR²⁸,    -   C₁-C₆-alkyl optionally substituted with one or more substituents        selected from halogen, —CN, —CF₃, —OCF₃, —OR²⁸, and —NR²⁸R²⁹    -   aryl, aryloxy, aroyl, aryl-C₁-C₆-alkoxy, aryl-C₁-C₆-alkyl,        heteroaryl, heteroaryl-C₁-C₆-alkyl,    -   of which the cyclic moieties optionally may be substituted with        one or more substituents selected from halogen, —C(O)OR²⁸, —CN,        —CF₃, —OCF₃, —NO₂, —OR²⁸, —NR²⁸R²⁹ and C₁-C₆-alkyl

In another embodiment of the invention R²⁵, R²⁶ and R²⁷ areindependently selected from

-   -   hydrogen, halogen, —OCF₃, —OR²⁸, —NR²⁸R²⁹, —SR²⁸, —NR²⁸C(O)R²⁹,        —NR²⁸C(O)OR²⁹, —OC(O)R²⁸, —OC₁-C₆-alkyl-C(O)OR²⁸,        —SC₁-C₆-alkyl-C(O)OR²⁸, —C₂-C₆-alkenyl-C(═O)OR²⁸,        —C(═O)NR²⁸—C₁-C₆-alkyl-C(═O)OR²⁸, —C₁-C₆-alkyl-C(═O)OR²⁸, or        —C(O)OR²⁸,    -   C₁-C₆-alkyl optionally substituted with one or more substituents        selected from halogen, —CN, or —CF₃    -   aryl, aryloxy, aroyl, aryl-C₁-C₆-alkoxy, aryl-C₁-C₆-alkyl,        heteroaryl, heteroaryl-C₁-C₆-alkyl,    -   of which the cyclic moieties optionally may be substituted with        one or more substituents selected from halogen, —C(O)OH, —CN,        —CF₃, —NO₂, or C₁-C₆-alkyl

In another embodiment of the invention R²¹ is hydrogen or methyl

In another embodiment of the invention R²¹ is hydrogen

In another embodiment of the invention R²⁸ is Hydrogen, C₁-C₆-alkyl oraryl

In another embodiment of the invention R²⁸ is Hydrogen or C₁-C₆-alkyl

In another embodiment of the invention R²⁹ is Hydrogen or C₁-C₆-alkyl

In another embodiment of the invention R¹⁸ and R¹⁹ are independentlyselected from

-   -   hydrogen, halogen, —CN, —CF₃, —OCF₃, —NO₂, —OR²⁸, —NR²⁸R²⁹,        —SR²⁸, —S(O)R²⁸, —S(O)₂R²⁸, —C(O)NR²⁸R²⁹, —CH₂OR²⁸, —OC(O)R²⁸,        —OC₁-C₆-alkyl-C(O)OR²⁸, —SC₁-C₆-alkyl-C(O)OR²⁸, or —C(O)OR²⁸,    -   C₁-C₆-alkyl, C₂-C₆-alkenyl or C₂-C₆-alkynyl,    -   which may optionally be substituted with one or more        substituents selected from halogen, —CN, —CF₃, —OCF₃, —OR²⁸, and        —NR²⁸R²⁹    -   aryl, aryloxy, aryl-C₁-C₆-alkoxy, aryl-C₁-C₆-alkyl, heteroaryl,        heteroaryl-C₁-C₆-alkyl of which the cyclic moieties optionally        may be substituted with one or more substituents selected from        halogen, —C(O)OR²⁸, —CN, —CF₃, —OCF₃, —NO₂, —OR²⁸, —NR²⁸R²⁹ and        C₁-C₆-alkyl

In another embodiment of the invention R¹⁸ and R¹⁹ are independentlyselected from

-   -   hydrogen, halogen, —CN, —CF₃, —NO₂, —OR²⁸, —NR²⁸R²⁹, or        —C(O)OR²⁸,    -   C₁-C₆-alkyl optionally substituted with one or more substituents        selected from halogen, —CN, —CF₃, —OCF₃, —OR²⁸, and —NR²⁸R²⁹    -   aryl, aryloxy, aryl-C₁-C₆-alkyl, heteroaryl, of which the cyclic        moieties optionally may be substituted with one or more        substituents selected from halogen, —C(O)OR²⁸, —CN, —CF₃, —OCF₃,        —NO₂, —OR²⁸, —NR²⁸R²⁹ and C₁-C₆-alkyl

In another embodiment of the invention A is

In another embodiment of the invention A is of the form M-Q-T-

-   -   wherein M is

-   -   wherein W¹, W², and W³ are independently OH, SH or NH₂ and the        phenyl, naphthalene or benzocarbazole rings are optionally        substituted by one or more R³⁴ independently    -   Q is selected from the following:        -   a valence bond        -   —CH₂N(R³⁰)— or —SO₂N(R³¹)—        -   A compound of the formula

-   -   -    wherein Z¹ is S(O)₂ or CH₂, Z² is N, —O— or —S—, and n is 1            or 2;

    -   T is        -   C₁-C₆-alkylene, C₂-C₆-alkenylene or C₂-C₆-alkynylene, which            may optionally be substituted with; one or more substituents            selected from halogen, —CN, —CF₃, —OCF₃, —OR³², and —NR³²R³³        -   Arylene, arylene-oxy, -aryl-oxycarbonyl-, -aroyl-,            -aryl-C₁-C₆-alkoxy-, -aryl-C₁-C₆-alkyl-,            -aryl-C₂-C₆-alkenyl-, -aryl-C₂-C₆-alkynyl-, heteroarylene,            -heteroaryl-C₁-C₆-alkyl-, -heteroaryl-C₂-C₆-alkenyl- or            -heteroaryl-C₂-C₆-alkynyl-, wherein the cyclic moieties are            optionally substituted by one or more substituents selected            from halogen, —C(O)OR³², —C(O)H, —CN, —CF₃, —OCF₃, —NO₂,            —OR³², —NR³²R³³, C₁-C₆-alkyl or C₁-C₆-alkanoyl,        -   A valence bond

    -   R³² and R³³ independently are hydrogen, C₁-C₆-alkyl,        aryl-C₁-C₆-alkyl or aryl, or R³² and R³³ when attached to the        same nitrogen atom together with the said nitrogen atom may form        a 3 to 8 membered heterocyclic ring optionally containing one or        two further heteroatoms selected from nitrogen, oxygen and        sulphur, and optionally containing one or two double bonds,

    -   R³⁰ and R³¹ are independently hydrogen, C₁-C₆-alkyl or        C₁-C₆-alkanoyl.

    -   R³⁴ is hydrogen, halogen, —CN, —CH₂CN, —CHF₂, —CF₃, —OCF₃,        —OCHF₂, —OCH₂CF₃, —OCF₂CHF₂, —S(O)₂CF₃, —SCF₃, —NO₂, —OR³²,        —C(O)R³², —NR³²R³³, —SR³², —NR³²S(O)₂R³³, —S(O)₂NR³²R³³,        —S(O)NR³²R³³, —S(O)R³², —S(O)₂R³², —C(O)NR³²R³³, —OC(O)NR³²R³³,        —NR³²C(O)R³³, —CH₂C(O)NR³²R³³, —OCH₂C(O)NR³²R³³, —CH₂OR³²,        —CH₂NR³²R³³, —OC(O)R³², —OC₁-C₆-alkyl-C(O)OR³²,        —SC₁-C₆-alkyl-C(O)OR³²—C₂-C₆-alkenyl-C(═O)OR³²,        —NR³²—C(═O)—C₁-C₆-alkyl-C(═O)OR³²,        —NR³²—C(═O)—C₁-C₆-alkenyl-C(═O)OR³²—, C₁-C₆-alkyl,        C₁-C₆-alkanoyl or —C(O)OR³²,

In another embodiment of the invention M is

In another embodiment of the invention M is

In another embodiment of the invention M is

In another embodiment of the invention M is

In another embodiment of the invention M is

In another embodiment of the invention Q is a valence bond, —CH₂N(R³⁰)—,or —SO₂N(R³¹)—

In another embodiment of the invention Q is a valence bond

In another embodiment of the invention T is

-   -   A valence bond    -   C₁-C₆-alkylene, C₂-C₆-alkenylene or C₂-C₆-alkynylene, which may        optionally be substituted with one or more substituents selected        from halogen, —CN, —CF₃, —OCF₃, —OR³², and —NR³²R³³    -   Arylene, or heteroarylene, wherein the cyclic moieties are        optionally substituted as defined in claim 70

In another embodiment of the invention T is

-   -   A valence bond    -   Arylene, or heteroarylene, wherein the cyclic moieties are        optionally substituted as defined in claim 70

In another embodiment of the invention T is phenylene or naphthalene

In another embodiment of the invention the cyclic moiety in T isoptionally substituted by halogen, —C(O)OR³², —CN, —CF₃, —OR³²,—NR³²R³³, C₁-C₆-alkyl or C₁-C₆-alkanoyl

In another embodiment of the invention the cyclic moiety in T isoptionally substituted by halogen, —C(O)OR³², —OR³², —NR³²R³³,C₁-C₆-alkyl or C₁-C₆-alkanoyl

In another embodiment of the invention the cyclic moiety in T isoptionally substituted by halogen, —C(O)OR³² or —OR³²

In another embodiment of the invention T is a valence bond

In another embodiment of the invention R³⁰ and R³¹ are independentlyhydrogen or C₁-C₆-alkyl

In another embodiment of the invention R³⁴ is hydrogen, halogen, —CN,—CF₃, —OCF₃, —SCF₃, —NO₂, —OR³², —C(O)R³², —NR³²R³³, —SR³²,—C(O)NR³²R³³, —OC(O)NR³²R³³, —NR³²C(O)R³³, —OC(O)R³²,—OC₁-C₆-alkyl-C(O)OR³², —SC₁-C₆-alkyl-C(O)OR³² or —C(O)OR³²

In another embodiment of the invention R³⁴ is hydrogen, halogen, —CF₃,—NO₂, —OR³², —NR³²R³³, —SR³², —NR³²C(O)R³³, or —C(O)OR³²

In another embodiment of the invention R³⁴ is hydrogen, halogen, —CF₃,—NO₂, —OR³², —NR³²R³³, or —NR³²C(O)R³³

In another embodiment of the invention R³⁴ is hydrogen, halogen, or—OR³²

In another embodiment of the invention R³² and R³³ independently arehydrogen, C₁-C₆-alkyl, or aryl

In another embodiment of the invention R³² and R³³ independently arehydrogen or C₁-C₆-alkyl

In another embodiment of the invention A is

-   -   wherein A¹ is a valence bond, C₁-C₆-alkylene, —NH—C(═O)-A²-,        —C₁-C₆-alkyl-S—, —C₁-C₆-alkyl-O—, —C(═O)—, or —C(═O)—NH—,        wherein any C₁-C₆-alkyl moiety is optionally substituted by        R^(1A);    -   A² is a valence bond, C₁-C₆-alkylene, C₁-C₆-alkenylene, or        —C₁-C₆-alkyl-O—;    -   R^(1A) is C₁-C₆-alkyl, aryl, wherein the alkyl or aryl moieties        are optionally substituted by one or more halogen, cyano, nitro,        amino;    -   AR¹ is a valence bond, arylene or heteroarylene, wherein the        aryl or heteroaryl moieties are optionally substituted by one or        more R^(1B) independently    -   R^(1B) is selected from        -   hydrogen, halogen, —CN, —CH₂CN, —CHF₂, —CF₃, —OCF₃, —OCHF₂,            —OCH₂CF₃, —OCF₂CHF₂, —S(O)₂CF₃, —OS(O)₂CF₃, —SCF₃, —NO₂,            —OR^(1C), —NR^(1C)R^(1D), —SR^(1C), —NR^(1C)S(O)₂R^(1D),            —S(O)₂NR^(1C)R^(1D), —S(O)NR^(1C)R^(1D), —S(O)R^(1C),            —S(O)₂R^(1C), —OS(O)₂R^(1C), —C(O)NR^(1C)R^(1D),            —OC(O)NR^(1C)R^(1D), —NR^(1C)C(O)R^(1D),            —CH₂C(O)NR^(1C)R^(1D), —OC₁-C₆-alkyl-C(O)NR^(1C)R^(1D),            —CH₂OR^(1C), —CH₂OC(O)R^(1C), —CH₂NR^(1C)R^(1D),            —OC(O)R^(1C), —OC₁-C₆-alkyl-C(O)OR^(1C),            —OC₁-C₆-alkyl-OR^(1C), —S—C₁-C₆-alkyl-C(O)OR^(1C),            —C₂-C₆-alkenyl-C(═O)OR^(1C),            —NR^(1C)—C(═O)—C₁-C₆-alkyl-C(═O)OR^(1C),            —NR^(1C)—C(═O)—C₁-C₆-alkenyl-C(═O)OR^(1C),            —C₂-C₆-alkenyl-C(═O)R^(1C), ═O, —NH—C(═O)—O—C₁-C₆-alkyl, or            —NH—C(═O)—C(═O)—O—C₁-C₆-alkyl        -   C₁-C₆-alkyl, C₂-C₆-alkenyl or C₂-C₆-alkynyl,        -   which may optionally be substituted with one or more            substituents selected from halogen, —CN, —CF₃, —OCF₃,            —OR^(1C), and —NR^(1C)R^(1D)        -   aryl, aryloxy, aryloxycarbonyl, arylsulfanyl,            aryl-C₁-C₆-alkoxy, aryl-C₁-C₆-alkyl, aryl-C₂-C₆-alkenyl,            aroyl-C₂-C₆-alkenyl, aryl-C₂-C₆-alkynyl, heteroaryl,            heteroaryl-C₁-C₆-alkyl, heteroaryl-C₂-C₆-alkenyl or            heteroaryl-C₂-C₆-alkynyl,        -   of which the cyclic moieties optionally may be substituted            with one or more substituents selected from halogen,            —C(O)OR^(1C), —CH₂C(O)OR^(1C), —CH₂OR^(1C), —CN, —CF₃,            —OCF₃, —NO₂, —OR^(1C), —NR^(1C)R^(1D) and C₁-C₆-alkyl,    -   R^(1C) and R^(1D) independently are hydrogen, —OH, C₁-C₆-alkyl,        C₁-C₆-alkenyl, aryl-C₁-C₆-alkyl or aryl, wherein the alkyl        moieties may optionally be substituted with one or more        substituents selected from halogen, —CN, —CF₃, —OCF₃,        —O—C₁-C₆-alkyl, —C(O)—O—C₁-C₆-alkyl, —COOH and —NH₂, and the        aryl moieties may optionally be substituted by halogen,        —C(O)OC₁-C₆-alkyl, —COOH, —CN, —CF₃, —OCF₃, —NO₂, —OH,        —OC₁-C₆-alkyl, —NH₂, C(═O) or C₁-C₆-alkyl; R^(1C) and R^(1D)        when attached to the same nitrogen atom may form a 3 to 8        membered heterocyclic ring with the said nitrogen atom, the        heterocyclic ring optionally containing one or two further        heteroatoms selected from nitrogen, oxygen and sulphur, and        optionally containing one or two double bonds,    -   C¹ is a valence bond, C₁-C₆-alkylene, —C₁-C₆-alkyl-O—,        —C₁-C₆-alkyl-NH—, —NH—C₁-C₆-alkyl, —NH—C(═O)—, —C(═O)—NH—,        —O—C₁-C₆-alkyl, —C(═O)—, or —C₁-C₆-alkyl-C(═O)—N(R^(1E)) wherein        the alkyl moieties are optionally substituted by one or more        R^(1F) independently    -   R^(1E) and R^(1F) are independently selected from C₁-C₆-alkyl,        aryl optionally substituted by one or more halogen, —COOH;    -   AR² is        -   a valence bond        -   C₁-C₆-alkylene, C₂-C₆-alkenylene, C₂-C₆-alkynylene wherein            the alkyl, alkenyl and alkynyl moieties are optionally            substituted by one or more R^(2A) independently;        -   arylene, -aryloxy-, -aryloxy-carbonyl-, aryl-C₁-C₆-alkyl,            -aroyl-, aryl-C₁-C₆-alkoxy-, aryl-C₂-C₆-alkenyl-,            aryl-C₂-C₆-alkynyl-, heteroarylene,            -heteroaryl-C₁-C₆-alkyl-, -heteroaryl-C₂-C₆-alkenyl-,            -heteroaryl-C₂-C₆-alkynyl- wherein the aryl and heteroaryl            moieties are optionally substituted by one or more R^(2A)            independently;    -   R^(2A) is C₁-C₆-alkyl, C₁-C₆-alkoxy, aryl, aryloxy,        aryl-C₁-C₆-alkoxy, —C(═O)—NH—C₁-C₆-alkyl-aryl, heteroaryl,        heteroaryl-C₁-C₆-alkoxy, —C₁-C₆-alkyl-COOH, —O—C₁-C₆-alkyl-COOH,        —S(O)₂R^(2B), —C₂-C₆-alkenyl-COOH, —OR^(2B), —NO₂, halogen,        —COOH, —CF₃, —CN, —N(R^(2B)R^(2C)), wherein the aryl or        heteroaryl moieties are optionally substituted by one or more        C₁-C₆-alkyl, C₁-C₆-alkoxy, —C₁-C₆-alkyl-COOH,        —C₂-C₆-alkenyl-COOH, —OR^(2B), —NO₂, halogen, —COOH, —CF₃, —CN,        or —N(R^(2B)R^(2C))    -   R^(2B) and R^(2C) are independently selected from hydrogen and        C₁-C₆-alkyl

In another embodiment of the invention A¹ is a valence bond,C₁-C₆-alkylene, —NH—C(═O)-A²-, —C₁-C₆-alkyl-S—, —C₁-C₆-alkyl-O—, or—C(═O)—, wherein any C₁-C₆-alkyl moiety is optionally substituted byR^(1A)

In another embodiment of the invention A¹ is a valence bond,C₁-C₆-alkylene, —NH—C(═O)-A²-, —C₁-C₆-alkyl-S—, or —C₁-C₆-alkyl-O,wherein any C₁-C₆-alkyl moiety is optionally substituted by R^(1A)

In another embodiment of the invention A¹ is a valence bond,C₁-C₆-alkylene, or —NH—C(═O)-A², wherein any C₁-C₆-alkyl moiety isoptionally substituted by R^(1A)

In another embodiment of the invention A¹ is a valence bond orC₁-C₆-alkylene, wherein any C₁-C₆-alkyl moiety is optionally substitutedby R^(1A)

In another embodiment of the invention A¹ is a valence bond

In another embodiment of the invention A² is a valence bond or—C₁-C₆-alkyl-O—

In another embodiment of the invention A² is a valence bond

In another embodiment of the invention AR¹ is arylene or heteroarylene,wherein the aryl or heteroaryl moieties are optionally substituted byone or more R^(1B) independently

In another embodiment of the invention AR¹ is selected from the group ofcompounds consisting of phenylene, biphenylylene, naphthylene,anthracenylene, phenanthrenylene, fluorenylene, indenylene, azulenylene,furylene, thienylene, pyrrolylene, oxazolylene, thiazolylene,imidazolylene, isoxazolylene, isothiazolylene, 1,2,3-triazolylene,1,2,4-triazolylene, pyranylene, pyridylene, pyridazinylene,pyrimidinylene, pyrazinylene, 1,2,3-triazinylene, 1,2,4-triazinylene,1,3,5-triazinylene, 1,2,3-oxadiazolylene, 1,2,4-oxadiazolylene,1,2,5-oxadiazolylene, 1,3,4-oxadiazolylene, 1,2,3-thiadiazolylene,1,2,4-thiadiazolylene, 1,2,5-thiadiazolylene, 1,3,4-thiadiazolylene,tetrazolylene, thiadiazinylene, indolylene, isoindolylene,benzofurylene, benzothienylene, indazolylene, benzimidazolylene,benzthiazolylene, benzisothiazolylene, benzoxazolylene,benzisoxazolylene, purinylene, quinazolinylene, quinolizinylene,quinolinylene, isoquinolinylene, quinoxalinylene, naphthyridinylene,pteridinylene, carbazolylene, azepinylene, diazepinylene, oracridinylene, optionally substituted by one or more R^(1B) independently

In another embodiment of the invention AR¹ is selected from phenylene,biphenylylene, naphthylene, pyridinylene, fyrylene, indolylene, orcarbazolylene, optionally substituted by one or more R^(1B)independently

In another embodiment of the invention AR¹ is selected from the group ofcompounds consisting of phenylene, indolylene, or carbazolylene,optionally substituted by one or more R_(1B) independently

In another embodiment of the invention AR¹ is phenylene optionallysubstituted by one or more R^(1B) independently

In another embodiment of the invention AR¹ is indolylene

In another embodiment of the invention AR¹ is carbazolylene

In another embodiment of the invention AR¹ is

In another embodiment of the invention AR¹ is

In another embodiment of the invention R^(1B) is selected from

-   -   hydrogen, halogen, —CN, —CF₃, —OCF₃, —NO₂, —OR^(1C),        —NR^(1C)R^(1D), —SR^(1C), —S(O)₂R^(1C), —NR^(1C)C(O)R^(1D),        —OC₁-C₆-alkyl-C(O)NR^(1C)R^(1D), —C₂-C₆-alkenyl-C(═O)OR^(1C),        —C(O)OR^(1C), ═O, —NH—C(═O)—O—C₁-C₆-alkyl, or        —NH—C(═O)—C(═O)—O—C₁-C₆-alkyl    -   C₁-C₆-alkyl or C₂-C₆-alkenyl    -   which may optionally be substituted with one or more        substituents selected from halogen, —CN, —CF₃, —OCF₃, —OR^(1C),        and —NR^(1C)R^(1D)    -   aryl, aryloxy, aryl-C₁-C₆-alkoxy, aryl-C₁-C₆-alkyl,        aryl-C₂-C₆-alkenyl, heteroaryl, heteroaryl-C₁-C₆-alkyl, or        heteroaryl-C₂-C₆-alkenyl        of which the cyclic moieties optionally may be substituted with        one or more substituents selected from halogen, —C(O)OR^(1C),        —CN, —CF₃, —OCF₃, —NO₂, —OR^(1C), —NR^(1C)R^(1D) and C₁-C₆-alkyl

In another embodiment of the invention R^(1B) is selected from

-   -   hydrogen, halogen, —CF₃, —NO₂, —OR^(1C), —NR^(1C)R^(1D),        —C(O)OR^(1C), ═O, —NH—C(═O)—O—C₁-C₆-alkyl, or        —NH—C(═O)—C(═O)—O—C₁-C₆-alkyl    -   C₁-C₆-alkyl

In another embodiment of the invention R^(1C) and R^(1D) independentlyare hydrogen, C₁-C₆-alkyl, or aryl, wherein the aryl moieties mayoptionally be substituted by halogen or —COOH

In another embodiment of the invention R^(1C) and R^(1D) independentlyare hydrogen, methyl, ethyl, or phenyl, wherein the phenyl moieties mayoptionally be substituted by halogen or —COOH

In another embodiment of the invention C¹ is a valence bond,C₁-C₆-alkylene, —C₁-C₆-alkyl-O-—, —C₁-C₆-alkyl-NH—, —NH—C₁-C₆-alkyl,—NH—C(═O)—, —C(═O)—NH—, —O—C₁-C₆-alkyl, —C(═O)—, or—C₁-C₆-alkyl-C(═O)—N(R^(1E))— wherein the alkyl moieties are optionallysubstituted by one or more R^(1F) independently

In another embodiment of the invention C¹ is a valence bond, —CH₂—,—CH₂—CH₂—, —CH₂—O—, —CH₂—CH₂—O—, —CH₂—NH—, —CH₂—CH₂—NH—, —NH—CH₂—,—NH—CH₂—CH₂—, —NH—C(═O)—, —C(═O)—NH—, —O—CH₂—, —O—CH₂—CH₂—, or —C(═O)—

In another embodiment of the invention R^(1E) and R^(1F) areindependently selected from C₁-C₆-alkyl

In another embodiment of the invention AR² is

-   -   a valence bond    -   C₁-C₆-alkylene, wherein the alkyl is optionally substituted by        one or more R^(2A) independently    -   arylene, aryl-C₁-C₆-alkyl, heteroarylene, wherein the aryl and        heteroaryl moieties are optionally substituted by one or more        R^(2A) independently

In another embodiment of the invention AR² is

-   -   a valence bond    -   C₁-C₆-alkylene, wherein the alkyl is optionally substituted by        one or more R^(2A) independently    -   phenyl, phenyl-C₁-C₆-alkyl, wherein the phenyl moieties are        optionally substituted by one or more R^(2A) independently

In another embodiment of the invention R^(2A) is C₁-C₆-alkyl,C₁-C₆-alkoxy, aryl, aryloxy, heteroaryl, —C₁-C₆-alkyl-COOH,—O—C₁-C₆-alkyl-COOH, —S(O)₂R^(2B), —C₂-C₆-alkenyl-COOH, —OR^(2B), —NO₂,halogen, —COOH, —CF₃, —CN, —N(R^(2B)R^(2C)), wherein the aryl orheteroaryl moieties are optionally substituted by one or moreC₁-C₆-alkyl, C₁-C₆-alkoxy, —C₁-C₆-alkyl-COOH, —C₂-C₆-alkenyl-COOH,—OR^(2B), —NO₂, halogen, —COOH, —CF₃, —CN, or —N(R^(2B)R^(2C))

In another embodiment of the invention R^(2A) is C₁-C₆-alkyl,C₁-C₆-alkoxy, aryl, —OR^(2B), —NO₂, halogen, —COOH, —CF₃, —CN,—N(R^(2B)R^(2C)), wherein the aryl is optionally substituted by one ormore C₁-C₆-alkyl, C₁-C₆-alkoxy, —OR^(2B), —NO₂, halogen, —COOH, —CF₃,—CN, or —N(R^(2B)R^(2C))

In another embodiment of the invention R^(2A) is C₁-C₆-alkyl,C₁-C₆-alkoxy, aryl, halogen, —CF₃, wherein the aryl is optionallysubstituted by one or more C₁-C₆-alkyl, halogen, —COOH, —CF₃, or —CN

In another embodiment of the invention R^(2A) is C₁-C₆-alkyl,C₁-C₆-alkoxy, phenyl, halogen, —COOH, —CF₃, or —CN

In another embodiment of the invention A is

-   -   wherein AR³ is C₁-C₆-alkylene, arylene, heteroarylene,        -aryl-C₁₋₆-alkyl- or -aryl-C₂₋₆-alkenyl-, wherein the alkylene        or alkenylene is optionally substituted with one or more        substituents independently selected from halogen, —CN, —CF₃,        —OCF₃, aryl, —COOH and —NH₂, and the arylene or heteroarylene is        optionally substituted with one or more R^(3A) independently    -   R^(3A) is independently selected from        -   hydrogen, halogen, —CN, —CH₂CN, —CHF₂, —CF₃, —OCF₃, —OCHF₂,            —OCH₂CF₃, —OCF₂CHF₂, —S(O)₂CF₃, —OS(O)₂CF₃, —SCF₃, —NO₂,            —OR^(3B), —NR^(3B)R^(3C), —SR^(3B), —NR^(3B)S(O)₂R^(3C),            —S(O)₂NR^(3B)R^(4C), —S(O)NR^(3B)R^(3C), —S(O)R^(3B),            —S(O)₂R^(3B), —OS(O)₂R^(3B), —C(O)NR^(3B)R^(3C),            —OC(O)NR^(3B)R^(3C), —NR^(3B)C(O)R^(3C),            —CH₂C(O)NR^(3C)R^(3B), —OC₁-C₆-alkyl-C(O)NR^(3B)R^(3C),            —CH₂OR^(3B), —CH₂OC(O)R^(3B), —CH₂NR^(3B)R^(3C),            —OC(O)R^(3B), —OC₁-C₆-alkyl-C(O)OR^(3B),            —OC₁-C₆-alkyl-OR^(3B), —SC₁-C₆-alkyl-C(O)OR^(3B),            —C₂-C₆-alkenyl-C(═O)OR^(3B),            —NR^(3B)—C(═O)—C₁-C₆-alkyl-C(═O)OR^(3B),            —NR^(3B)—C(═O)—C₁-C₆-alkenyl-C(═O)OR^(3B), —C(O)OR^(3B), or            —C₂-C₆-alkenyl-C(═O)R^(3B),        -   C₁-C₆-alkyl, C₂-C₆-alkenyl or C₂-C₆-alkynyl,        -   which may optionally be substituted with one or more            substituents selected from halogen, —CN, —CF₃, —OCF₃,            —OR^(3B), and —NR^(3B)R^(3C)        -   aryl, aryloxy, aryloxycarbonyl, aroyl, arylsulfanyl,            aryl-C₁-C₆-alkoxy, aryl-C₁-C₆-alkyl, aryl-C₂-C₆-alkenyl,            aroyl-C₂-C₆-alkenyl, aryl-C₂-C₆-alkynyl, heteroaryl,            heteroaryl-C₁-C₆-alkyl, heteroaryl-C₂-C₆-alkenyl or            heteroaryl-C₂-C₆-alkynyl,        -   of which the cyclic moieties optionally may be substituted            with one or more substituents selected from halogen,            —C(O)OR^(3B), —CH₂C(O)OR^(3B), —CH₂OR^(3B), —CN, —CF₃,            —OCF₃, —NO₂, —OR^(3B), —NR^(3B)R^(3C) and C₁-C₆-alkyl,    -   R^(3B) and R^(3C) are independently hydrogen, OH, CF₃,        C₁-C₂-alkyl, aryl-C₁-C₆-alkyl, —C(═O)—C₁-C₆-alkyl or aryl,        wherein the alkyl groups may optionally be substituted with one        or more substituents selected from halogen, —CN, —CF₃, —OCF₃,        —OC₁-C₆-alkyl, —C(O)OC₁-C₆-alkyl, —C(═O)—R^(3D), —COOH and —NH₂,        and the aryl groups may optionally be substituted by halogen,        —C(O)OC₁-C₆-alkyl, —COOH, —CN, —CF₃, —OCF₃, —NO₂, —OH,        —OC₁-C₆-alkyl, —NH₂, C(═O) or C₁-C₆-alkyl; R^(3B) and R^(3C)        when attached to the same nitrogen atom may form a 3 to 8        membered heterocyclic ring with the said nitrogen atom, the        heterocyclic ring optionally containing one or two further        heteroatoms selected from nitrogen, oxygen and sulphur, and        optionally containing one or two double bonds    -   R^(3D) is C₁-C₆-alkyl, aryl optionally substituted with one or        more halogen, or heteroaryl optionally substituted with one or        more C₁-C₆-alkyl.

In another embodiment of the invention AR³ is arylene, heteroarylene, oraryl-C₁₋₆-alkyl, wherein the alkyl is optionally substituted with one ormore substituents independently selected from halogen, —CN, —CF₃, —OCF₃,aryl, —COOH and —NH₂, and the arylene or heteroarylene is optionallysubstituted with one or more R^(3A) independently

In another embodiment of the invention AR³ is arylene optionallysubstituted with one or more R^(3A) independently

In another embodiment of the invention AR³ is phenylene, naphthalene oranthranylene optionally substituted with one or more R^(3A)independently

In another embodiment of the invention AR³ is phenylene optionallysubstituted with one or more R^(3A) independently

In another embodiment of the invention R^(3A) is independently selectedfrom

-   -   halogen, —CN, —CF₃, —NO₂, —OR^(3B), —NR^(3B)R^(3C), —SR^(3B),        —OC₁-C₆-alkyl-C(O)OR^(3B) or —C(O)OR^(3B)    -   C₁-C₆-alkyl optionally substituted with one or more substituents        selected from halo gen, —CN, —CF₃, —OCF₃, —OR^(3B), and        —NR^(3B)R^(3C)    -   aryl, aryl-C₁-C₆-alkyl, heteroaryl, or heteroaryl-C₁-C₆-alkyl of        which the cyclic moieties optionally may be substituted with one        or more substituents selected from halogen, —C(O)OR^(3B), —CN,        —CF₃, —OCF₃, —NO₂, —OR^(3B), —NR^(3B)R^(3C) and C₁-C₆-alkyl

In another embodiment of the invention R^(3A) is independently selectedfrom halogen, —OR^(3B), —NR^(3B)R^(3C), —C(O)OR^(3B),—OC₁-C₆-alkyl-C(O)OR^(3B), or C₁-C₆-alkyl

In another embodiment of the invention R^(3B) and R^(3C) areindependently hydrogen, CF₃, C₁-C₁₂-alkyl, or —C(═O)—C₁-C₆-alkyl; R^(3B)and R^(3C) when attached to the same nitrogen atom may form a 3 to 8membered heterocyclic ring with the said nitrogen atom

In another embodiment of the invention A is

-   -   wherein AR⁴ is C₁-C₆-alkylene, arylene, heteroarylene,        -aryl-C₁₋₆-alkyl- or -aryl-C₂₋₆-alkenyl, wherein the alkylene or        alkenylene is optionally substituted with one or more        substituents independently selected from halogen, —CN, —CF₃,        —OCF₃, aryl, —COOH and —NH₂, and the arylene or heteroarylene is        optionally substituted with one or more R^(4A) independently    -   R^(4A) is independently selected from        -   hydrogen, halogen, —CN, —CH₂CN, —CHF₂, —CF₃, —OCF₃, —OCHF₂,            —OCH₂CF₃, —OCF₂CHF₂, —S(O)₂CF₃, —OS(O)₂CF₃, —SCF₃, —NO₂,            —OR^(4B), —NR^(4B)R^(4C), —SR^(4B), —NR^(4B)S(O)₂R^(4C),            —S(O)₂NR^(4B)R^(4C), —S(O)NR^(4B)R^(4C), —S(O)R^(4B),            —S(O)R^(4B), —OS(O)₂R^(4B), —C(O)NR^(4B)R^(4C),            —OC(O)NR^(4B)R^(4C), —NR^(4B)C(O)R^(4C),            —CH₂C(O)NR^(4B)R^(4C), —OC₁-C₆-alkyl-C(O)NR^(4B)R^(4C),            —CH₂OR^(4B), —CH₂OC(O)R^(4B), —CH₂NR^(4B)R^(4C),            —OC(O)R^(4B), —OC₁-C₆-alkyl-C(O)O^(4B),            —OC₁-C₆-alkyl-OR^(4B), —SC₁-C₆-alkyl-C(O)OR^(4B),            —C₂-C₆-alkenyl-C(═O)OR^(4B),            —NR^(4B)—C(═O)—C₁-C₆-alkyl-C(═O)OR^(4B),            —NR^(4B)—C(═O)—C₁-C₆-alkenyl-C(═O)OR^(4B), —C(O)OR^(4B), or            —C₂-C₆-alkenyl-C(═O)R^(4B),        -   C₁-C₆-alkyl, C₂-C₆-alkenyl or C₂-C₆-alkynyl,        -   which may optionally be substituted with one or more            substituents selected from halogen, —CN, —CF₃, —OCF₃,            —OR^(4B), and —NR^(4B)R^(4C)        -   aryl, aryloxy, aryloxycarbonyl, aroyl, arylsulfanyl,            aryl-C₁-C₆-alkoxy, aryl-C₁-C₆-alkyl, aryl-C₂-C₆-alkenyl,            aroyl-C₂-C₆-alkenyl, aryl-C₂-C₆-alkynyl, heteroaryl,            heteroaryl-C₁-C₆-alkyl, heteroaryl-C₂-C₆-alkenyl or            heteroaryl-C₂-C₆-alkynyl,        -   of which the cyclic moieties optionally may be substituted            with one or more substituents selected from halogen,            —C(O)OR^(4B), —CH₂C(O)OR^(4B), —CH₂OR^(4B), —CN, —CF₃,            —OCF₃, —NO₂, —OR^(4B), —NR^(4B)R^(4C) and C₁-C₆-alkyl,    -   R^(4B) and R^(4C) are independently hydrogen, OH, CF₃,        C₁-C₁₂-alkyl, aryl-C₁-C₆-alkyl, —C(═O)—R^(4D), or aryl, wherein        the alkyl groups may optionally be substituted with one or more        substituents selected from halogen, —CN, —CF₃, —OCF₃,        —OC₁-C₆-alkyl, —C(O)OC₁-C₆-alkyl, —COOH and —NH₂, and the aryl        groups may optionally be substituted by halogen,        —C(O)OC₁-C₆-alkyl, —COOH, —CN, —CF₃, —OCF₃, —NO₂, —OH,        —OC₁-C₆-alkyl, —NH₂, C(═O) or C₁-C₆-alkyl; R^(4B) and R^(4C)        when attached to the same nitrogen atom may form a 3 to 8        membered heterocyclic ring with the said nitrogen atom, the        heterocyclic ring optionally containing one or two further        heteroatoms selected from nitrogen, oxygen and sulphur, and        optionally containing one or two double bonds    -   R^(4D) is C₁-C₆-alkyl, aryl optionally substituted with one or        more halogen, or heteroaryl optionally substituted with one or        more C₁-C₆-alkyl.

In another embodiment of the invention AR⁴ is arylene, heteroarylene oraryl-C₁₋₆-alkyl-, wherein the alkyl is optionally substituted with oneor more substituents independently selected from halogen, —CN, —CF₃,—OCF₃, aryl, —COOH and —NH₂, and the arylene or heteroaryl is optionallysubstituted with one or more R^(4A) independently

In another embodiment of the invention AR⁴ is arylene or heteroaryleneoptionally substituted with one or more R^(4A) independently

In another embodiment of the invention AR⁴ is phenylene, naphtylene,anthrylene, thienylene, pyridylene, or benzodioxylene optionallysubstituted with one or more R^(4A) independently

In another embodiment of the invention AR⁴ is phenylene optionallysubstituted with one or more R^(4A) independently

In another embodiment of the invention R^(4A) is independently selectedfrom hydrogen, halogen, —CF₃, —OR^(4B), —NR^(4B)R^(4C), C₁-C₆-alkyl,aryl-C₂-C₆-alkenyl or aryl optionally substituted with one or moresubstituents selected from halogen, —CF₃, or —OR^(4B)

In another embodiment of the invention R^(4B) and R^(4C) areindependently hydrogen, CF₃, C₁-C₁₂-alkyl, —C(═O)—R^(4D), or aryl

In another embodiment of the invention R^(4D) is C₁-C₆-alkyl, phenyloptionally substituted with one or more halogen, or a heteroarylselected from isoxazole and thiadiazole optionally substituted with oneor more C₁-C₆-alkyl

In another embodiment of the invention C consists of 0 to 5 neutralamino acids independently selected from the group consisting of Abz,Gly, Ala, Thr, and Ser

In another embodiment of the invention C consists of 0 to 5 Gly

In another embodiment of the invention C consists of 0 Gly

In another embodiment of the invention C consists of 1 Gly

In another embodiment of the invention C consists of 2 Gly

In another embodiment of the invention C consists of 3 Gly

In another embodiment of the invention C consists of 4 Gly

In another embodiment of the invention C consists of 5 Gly

In another embodiment of the invention G^(B) is of the formula—B¹—B²—C(O)—, —B¹—B²—SO₂— or —B¹—B²—CH₂—, wherein B¹ and B² are asdefined in claim 1

In another embodiment of the invention G^(B) is of the formula—B¹—B²—C(O)—, —B¹—B²—SO₂— or —B¹—B²—NH—, wherein B¹ and B² are asdefined in claim 1

In another embodiment of the invention G^(B) is of the formula—B¹—B²—C(O)—, —B¹—B²—CH₂, or —B¹—B²—NH—, wherein B¹ and B² are asdefined in claim 1

In another embodiment of the invention G^(B) is of the formula—B¹—B²—CH₂—, —B¹—B²—SO₂— or —B¹—B²—NH—, wherein B¹ and B² are as definedin claim 1

In another embodiment of the invention G^(B) is of the formula—B¹—B²—C(O)— or —B¹—B²—SO₂—, wherein B¹ and B² are as defined in claim 1

In another embodiment of the invention G^(B) is of the formula—B¹—B²—C(O)— or —B¹—B²—CH₂—, wherein B¹ and B² are as defined in claim 1

In another embodiment of the invention G^(B) is of the formula—B¹—B²—C(O)— or —B¹—B²—NH—, wherein B¹ and B² are as defined in claim 1

In another embodiment of the invention G^(B) is of the formula—B¹—B²—CH₂— or —B¹—B²—SO₂—, wherein B¹ and B² are as defined in claim 1

In another embodiment of the invention G^(B) is of the formula—B¹—B²—NH— or —B¹—B²—SO₂—, wherein B¹ and B² are as defined in claim 1

In another embodiment of the invention G^(B) is of the formula—B¹—B²—CH₂— or —B¹—B²—NH—, wherein B¹ and B² are as defined in claim 1

In another embodiment of the invention G^(B) is of the formula—B¹—B²—C(O)—

In another embodiment of the invention G^(B) is of the formula—B¹—B²—CH₂—

In another embodiment of the invention G^(B) is of the formula—B¹—B²—SO₂—

In another embodiment of the invention G^(B) is of the formula—B¹—B²—NH—

In another embodiment of the invention B¹ is a valence bond, —O—, or —S—

In another embodiment of the invention B¹ is a valence bond, —O—, or—N(R⁶)—

In another embodiment of the invention B¹ is a valence bond, —S—, or—N(R⁶)—

In another embodiment of the invention B¹ is —O—, —S— or —N(R₆)—

In another embodiment of the invention B¹ is a valence bond —O—

In another embodiment of the invention B¹ is a valence bond or —S—

In another embodiment of the invention B¹ is a valence bond or —N(R⁶)—

In another embodiment of the invention B¹ is —O— or —S—

In another embodiment of the invention B¹ is —O— or —N(R⁶)—

In another embodiment of the invention B¹ is —S— or —N(R⁶)—

In another embodiment of the invention B¹ is a valence bond

In another embodiment of the invention B¹ is —O—

In another embodiment of the invention B¹ is —S—

In another embodiment of the invention B¹ is —N(R⁶)—

In another embodiment of the invention B² is a valence bond,C₁-C₁₈-alkylene, C₂-C₁₈-alkenylene, C₂-C₁₈-alkynylene, arylene,heteroarylene, —C₁-C₁₈-alkyl-aryl-, —C(═O)—C₁-C₁₈-alkyl-C(═O)—,—C(═O)—C₁-C₁₈-alkyl-O—C₁-C₁₈-alkyl-C(═O)—,—C(═O)—C₁-C₁₈-alkyl-S—C₁-C₁₈—C(═O)—,—C(═O)—C₁-C₁₈-alkyl-NR⁶—C₁-C₁₈-alkyl-C(═O)—; and the alkylene andarylene moieties are optionally substituted as defined in claim 1

In another embodiment of the invention B² is a valence bond,C₁-C₁₈-alkylene, C₂-C₁₈-alkenylene, C₂-C₁₈-alkynylene, arylene,heteroarylene, —C₁-C₁₈-alkyl-aryl-, —C(═O)—C₁-C₁₈-alkyl-C(═O)—,—C(═O)—C₁-C₁₈-alkyl-O—C₁-C₁₈-alkyl-C(═O)—, and the alkylene and arylenemoieties are optionally substituted as defined in claim 1

In another embodiment of the invention B² is a valence bond,C₁-C₁₈-alkylene, C₂-C₁₈-alkenylene, C₂-C₁₈-alkynylene, arylene,heteroarylene, —C₁-C₁₈-alkyl-aryl-, —C(═O)—C₁-C₁₈-alkyl-C(═O)—, and thealkylene and arylene moieties are optionally substituted as defined inclaim 1

In another embodiment of the invention B² is a valence bond,C₁-C₁₈-alkylene, arylene, heteroarylene, —C₁-C₁₈-alkyl-aryl-,—C(═O)—C₁-C₁₈-alkyl-C(═O)—, and the alkylene and arylene moieties areoptionally substituted as defined in claim 1

In another embodiment of the invention B² is a valence bond,C₁-C₁₈-alkylene, arylene, heteroarylene, —C₁-C₁₈-alkyl-aryl-, and thealkylene and arylene moieties are optionally substituted as defined inclaim 1

In another embodiment of the invention B² is a valence bond,C₁-C₁₈-alkylene, arylene, —C₁-C₁₈-alkyl-aryl-, and the alkylene andarylene moieties are optionally substituted as defined in claim 1

In another embodiment of the invention B² is a valence bond or—C₁-C₁₈-alkylene, and the alkylene moieties are optionally substitutedas defined in claim 1

In another embodiment of the invention D comprises 1 to 16 positivelycharged groups

In another embodiment of the invention D comprises 1 to 12 positivelycharged groups

In another embodiment of the invention D comprises 1 to 10 positivelycharged groups

In another embodiment of the invention D is a fragment containing basicamino acids independently selected from the group consisting of Lys andArg and D-isomers of these.

In another embodiment of the invention the basic amino acid is Arg

In another embodiment of the invention X is —OH or —NH₂

In another embodiment of the invention X is —NH₂

The invention furthermore provides an R-state insulin hexamercomprising:

-   -   6 molecules of insulin, at least 2 zinc ions, and a zinc-binding        ligand as defined above

In another embodiment of the invention the insulin is selected from thegroup consisting of human insulin, an analogue thereof, a derivativethereof, and combinations of any of these

In another embodiment of the invention the insulin is an analogue ofhuman insulin selected from the group consisting of

-   -   iii. An analogue wherein position B28 is Asp, Lys, Leu, Val, or        Ala and position B29 is Lys or Pro; and    -   iv. des(B28-B30), des(B27) or des(B30) human insulin.

In another embodiment of the invention the insulin is an analogue ofhuman insulin wherein position B28 is Asp or Lys, and position B29 isLys or Pro.

In another embodiment of the invention the insulin is des(B30) humaninsulin.

In another embodiment of the invention the insulin is a derivative ofhuman insulin having one or more lipophilic substituents.

In another embodiment of the invention the insulin derivative isselected from the group consisting of B29-N^(ε)-myristoyl-des(B30) humaninsulin, B29-N^(ε)-palmitoyl-des(B30) human insulin, B29-N^(ε)-myristoylhuman insulin, B29-N^(ε)-palmitoyl human insulin, B28-N^(ε)-myristoylLys^(B28) Pro^(B29) human insulin, B28-N^(ε)-palmitoyl Lys^(B28)Pro^(B29) human insulin, B30-N^(ε)-myristoyl-Thr^(B29)Lys^(B30) humaninsulin, B30-N^(ε)-palmitoyl-Thr^(B29)Lys^(B30) human insulin,B29-N^(ε)-(N-palmitoyl-γ-glutamyl)-des(B30) human insulin,B29-N^(ε)-(N-lithocholyl-γ-glutamyl)-des(B30) human insulin,B29-N^(ε)-(ω-carboxyheptadecanoyl)-des(B30) human insulin andB29-N^(ε)-(ω-carboxyheptadecanoyl) human insulin.

In another embodiment of the invention the insulin derivative isB29-N^(ε)-myristoyl-des(B30) human insulin.

In another embodiment of the invention the insulin hexamer as definedabove further comprises at least 3 phenolic molecules.

The invention furthermore provides an aqueous insulin preparationcomprising R-state insulin hexamers as defined above

The invention furthermore provides a method of prolonging the action ofan insulin preparation which comprises adding a zinc-binding ligand asdefined above to the insulin preparation.

In another embodiment of the invention the ratio between precipitatedinsulin and dissolved insulin is in the range from 99:1 to 1:99.

In another embodiment of the invention the ratio between precipitatedinsulin and dissolved insulin is in the range from 95:5 to 5:95

In another embodiment of the invention the ratio between precipitatedinsulin and dissolved insulin is in the range from 80:20 to 20:80

In another embodiment of the invention the ratio between precipitatedinsulin and dissolved insulin is in the range from 70:30 to 30:70

The invention furthermore provides a method of preparing a zinc-bindingligand as defined above comprising the steps of

-   -   Identifying starter compounds that are able to displace a ligand        from the R-state His^(B10)-Zn²⁺ site    -   optionally attaching a fragment consisting of 0 to 5 neutral α-        or β-amino acids    -   attaching a fragment comprising 1 to 20 positively charged        groups independently selected from amino or guanidino groups

The compounds of the present invention may be chiral, and it is intendedthat any enantiomers, as separated, pure or partially purifiedenantiomers or racemic mixtures thereof are included within the scope ofthe invention.

Furthermore, when a double bond or a fully or partially saturated ringsystem or more than one centre of asymmetry or a bond with restrictedrotatability is present in the molecule diastereomers may be formed. Itis intended that any diastereomers, as separated, pure or partiallypurified diastereomers or mixtures thereof are included within the scopeof the invention.

Furthermore, some of the compounds of the present invention may exist indifferent tautomeric forms and it is intended that any tautomeric forms,which the compounds are able to form, are included within the scope ofthe present invention.

The present invention also encompasses pharmaceutically acceptable saltsof the present compounds. Such salts include pharmaceutically acceptableacid addition salts, pharmaceutically acceptable metal salts, ammoniumand alkylated ammonium salts. Acid addition salts include salts ofinorganic acids as well as organic acids. Representative examples ofsuitable inorganic acids include hydrochloric, hydrobromic, hydroiodic,phosphoric, sulphuric, nitric acids and the like. Representativeexamples of suitable organic acids include formic, acetic,trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric,fumaric, glycolic, lactic, maleic, malic, malonic, mandelic, picric,pyruvic, succinic, methanesulfonic, ethanesulfonic, tartaric, ascorbic,pamoic, ethanedisulfonic, gluconic, citraconic, aspartic, stearic,palmitic, glycolic, p-aminobenzoic, glutamic, benzenesulfonic,p-toluenesulfonic acids and the like. Further examples ofpharmaceutically acceptable inorganic or organic acid addition saltsinclude the pharmaceutically acceptable salts listed in J. Pharm. Sci.1977, 66, 2, which is incorporated herein by reference. Examples ofmetal salts include lithium, sodium, potassium, magnesium salts and thelike. Examples of ammonium and alkylated ammonium salts includeammonium, methyl-, dimethyl-, trimethyl-, ethyl-, hydroxyethyl-,diethyl-, n-butyl-, sec-butyl-, tert-butyl-, tetramethylammonium saltsand the like.

Also intended as pharmaceutically acceptable acid addition salts are thehydrates, which the present compounds, are able to form.

Furthermore, the pharmaceutically acceptable salts comprise basic aminoacid salts such as lysine, arginine and ornithine.

The acid addition salts may be obtained as the direct products ofcompound synthesis. In the alternative, the free base may be dissolvedin a suitable solvent containing the appropriate acid, and the saltisolated by evaporating the solvent or otherwise separating the salt andsolvent.

The compounds of the present invention may form solvates with standardlow molecular weight solvents using methods well known to the personskilled in the art. Such solvates are also contemplated as being withinthe scope of the present invention.

Pharmaceutical Compositions

The present invention also relates to a pharmaceutical composition forthe treatment of diabetes in a patient in need of such a treatmentcomprising an R-state hexamer of insulin according to the inventiontogether with a pharmaceutically acceptable carrier.

In one embodiment of the invention the insulin preparation comprises 60to 3000 nmol/ml of insulin.

In another embodiment of the invention the insulin preparation comprises240 to 1200 nmol/ml of insulin.

In another embodiment of the invention the insulin preparation comprisesabout 600 nmol/ml of insulin.

Zinc ions may be present in an amount corresponding to 10 to 40 μgZn/100 U insulin, more preferably 10 to 26 μg Zn/100 U insulin.

Insulin formulations of the invention are usually administered frommulti-dose containers where a preservative effect is desired. Sincephenolic preservatives also stabilize the R-state hexamer theformulations may contain up to 50 mM of phenolic molecules. The phenolicmolecules in the insulin formulation may be selected from the groupconsisting of phenol, m-cresol, chloro-cresol, thymol, 7-hydroxyindoleor any mixture thereof.

In one embodiment of the invention 0.5 to 4.0 mg/ml of phenolic compoundmay be employed.

In another embodiment of the invention 0.6 to 4.0 mg/ml of m-cresol maybe employed.

In another embodiment of the invention 0.5 to 4.0 mg/ml of phenol may beemployed.

In another embodiment of the invention 1.4 to 4.0 mg/ml of phenol may beemployed.

In another embodiment of the invention 0.5 to 4.0 mg/ml of a mixture ofm-cresol or phenol may be employed.

In another embodiment of the invention 1.4 to 4.0 mg/ml of a mixture ofm-cresol or phenol may be employed.

The pharmaceutical preparation may further comprises a buffer substance,such as a TRIS, phosphate, glycine or glycylglycine (or anotherzwitterionic substance) buffer, an isotonicity agent, such as NaCl,glycerol, mannitol and/or lactose. Chloride would be used at moderateconcentrations (e.g. up to 50 mM) to avoid competition with thezinc-site ligands of the present invention.

The action of insulin may further be slowed down in vivo by the additionof physiologically acceptable agents that increase the viscosity of thepharmaceutical preparation. Thus, the pharmaceutical preparationaccording to the invention may furthermore comprise an agent whichincreases the viscosity, such as polyethylene glycol, polypropyleneglycol, copolymers thereof, dextrans and/or polylactides.

In a particular embodiment the insulin preparation of the inventioncomprises between 0.001% by weight and 1% by weight of anon-ionicsurfactant, for example tween 20 or Polox 188.

A nonionic detergent can be added to stabilise insulin againstfibrillation during storage and handling.

The insulin preparation of the present invention may have a pH value inthe range of 3.5 to 8.5, more preferably 7.4 to 7.9.

EXAMPLES

The following examples and general procedures refer to intermediatecompounds and final products identified in the specification and in thesynthesis schemes. The preparation of the compounds of the presentinvention is described in detail using the following examples, but thechemical reactions described are disclosed in terms of their generalapplicability to the preparation of compounds of the invention.Occasionally, the reaction may not be applicable as described to eachcompound included within the disclosed scope of the invention. Thecompounds for which this occurs will be readily recognised by thoseskilled in the art. In these cases the reactions can be successfullyperformed by conventional modifications known to those skilled in theart, that is, by appropriate protection of interfering groups, bychanging to other conventional reagents, or by routine modification ofreaction conditions. Alternatively, other reactions disclosed herein orotherwise conventional will be applicable to the preparation of thecorresponding compounds of the invention. In all preparative methods,all starting materials are known or may easily be prepared from knownstarting materials. All temperatures are set forth in degrees Celsiusand unless otherwise indicated, all parts and percentages are by weightwhen referring to yields and all parts are by volume when referring tosolvents and eluents.

HPLC-MS (Method A)

The following instrumentation was used:

-   -   Hewlett Packard series 1100 G1312A Bin Pump    -   Hewlett Packard series 1100 Column compartment    -   Hewlett Packard series 1100 G13 15A DAD diode array detector    -   Hewlett Packard series 1100 MSD

The instrument was controlled by HP Chemstation software.

The HPLC pump was connected to two eluent reservoirs containing:

-   -   A: 0.01% in water    -   B: 0.01% TFA in acetonitrile

The analysis was performed at 40° C. by injecting an appropriate volumeof the sample (preferably 1 μL) onto the column, which was eluted with agradient of acetonitrile.

The HPLC conditions, detector settings and mass spectrometer settingsused are given in the following table.

Column Waters Xterra MS C-18 × 3 mm id Gradient 10%-100% acetonitrilelineary during 7.5 min at 1.0 mL/min Detection UV: 210 nm (analog outputfrom DAD) MS Ionisation mode: API-ES Scan 100-1000 amu step 0.1 amuHPLC-MS (Method B)

The following instrumentation was used:

-   -   Sciex API 100 Single quadropole mass spectrometer    -   Perkin Elmer Series 200 Quard pump    -   Perkin Elmer Series 200 autosampler    -   Applied Biosystems 785A UV detector    -   Sedex 55 evaporative light scattering detector    -   A Valco column switch with a Valco actuator controlled by timed        events from the pump.

The Sciex Sample control software running on a Macintosh PowerPC 7200Computer was used for the instrument control and data acquisition.

The HPLC pump was connected to four eluent reservoirs containing:

-   -   A: acetonitrile    -   B: water    -   C: 0.5% TFA in water    -   D: 0.02 M ammonium acetate

The requirements for samples are that they contain approximately 500μg/mL of the compound to be analysed in an acceptable solvent such asmethanol, ethanol, acetonitrile, THF, water and mixtures thereof. (Highconcentrations of strongly eluting solvents will interfere with thechromatography at low acetonitrile concentrations.)

The analysis was performed at room temperature by injecting 20 μL of thesample solution on the column, which was eluted with a gradient ofacetonitrile in either 0.05% TFA or 0.002 M ammonium acetate. Dependingon the analysis method varying elution conditions were used.

The eluate from the column was passed through a flow splittingT-connector, which passed approximately 20 μL/min through approx. 1 m.75μ fused silica capillary to the API interface of API 100 spectrometer.

The remaining 1.48 mL/min was passed through the UV detector and to theELS detector.

During the LC-analysis the detection data were acquired concurrentlyfrom the mass spectrometer, the UV detector and the ELS detector.

The LC conditions, detector settings and mass spectrometer settings usedfor the different methods are given in the following table.

Column YMC ODS-A 120 Å s - 5μ 3 mm × 50 mm id Gradient 5%-90%acetonitrile in 0.05% TFA linearly during 7.5 min at 1.5 mL/minDetection UV: 214 nm ELS: 40° C. MS Experiment: Start: 100 amu Stop: 800amu Step: 0.2 amu Dwell: 0.571 msec Method: Scan 284 times = 9.5 minHPLC-MS (Method C)

The Following Instrumentation is Used:

-   -   Hewlett Packard series 1100 G1312A Bin Pump    -   Hewlett Packard series 1100 Column compartment    -   Hewlett Packard series 1100 G1315A DAD diode array detector    -   Hewlett Packard series 1100 MSD    -   Sedere 75 Evaporative Light Scattering detector

The instrument is controlled by HP Chemstation software.

The HPLC pump is connected to two eluent reservoirs containing:

A 0.01% TFA in water B 0.01% TFA in acetonitrile

The analysis is performed at 40° C. by injecting an appropriate volumeof the sample (preferably 1 μl) onto the column which is eluted with agradient of acetonitrile.

The HPLC conditions, detector settings and mass spectrometer settingsused are given in the following table.

Column Waters Xterra MS C-18 × 3 mm id 5 μm Gradient 50%-100%acetonitrile linear during 7.5 min at 1.5 ml/min Detection 210 nm(analogue output from DAD) ELS (analogue output from ELS) MS ionisationmode API-ES Scan 100-1000 amu step 0.1 amu

After the DAD the flow is divided yielding approximately 1 ml/min to theELS and 0.5 ml/min to the MS.

HPLC-MS (Method D)

The following instrumentation was used:

-   -   Sciex API 150 Single Quadropole mass spectrometer    -   Hewlett Packard Series 1100 G1312A Bin pump    -   Gilson 215 micro injector    -   Hewlett Packard Series 1100 G1315A DAD diode array detector    -   Sedex 55 evaporative light scattering detector    -   A Valco column switch with a Valco actuator controlled by timed        events from the pump.

The Sciex Sample control software running on a Macintosh Power G3Computer was used for the instrument control and data acquisition.

The HPLC pump was connected to two eluent reservoirs containing:

-   -   A: Acetonitrile containing 0.05% TFA    -   B: Water containing 0.05% TFA

The requirements for the samples are that they contain approximately 500μg/ml of the compound to be analysed in an acceptable solvent such asmethanol, ethanol, acetonitrile, THF, water and mixtures thereof. (Highconcentrations of strongly eluting solvents will interfere with thechromatography at low acetonitrile concentrations.)

The analysis was performed at room temperature by injecting 20 μl of thesample solution on the column, which was eluted with a gradient ofacetonitrile in 0.05% TFA The eluate from the column was passed througha flow splitting T-connector, which passed approximately 20 μl/minthrough approx. 1 m 75μ fused silica capillary to the API interface ofAPI 150 spectrometer.

The remaining 1.48 ml/min was passed through the UV detector and to theELS detector. During the LC-analysis the detection data were acquiredconcurrently from the mass spectrometer, the UV detector and the ELSdetector.

The LC conditions, detector settings and mass spectrometer settings usedfor the different methods are given in the following table.

Column Waters X-terra C18 5μ 3 mm × 50 mm id Gradient 5%-90%acetonitrile in 0.05% TFA linearly during 7.5 min at 1.5 ml/minDetection UV: 214 nm ELS: 40° C. MS Experiment: Start: 100 amu Stop: 800amu Step: 0.2 amu Dwell: 0.571 msec Method: Scan 284 times = 9.5 min

EXAMPLES Example 1 1H-Benzotriazole

Example 2 5,6-Dimethyl-1H-benzotriazole

Example 3 1H-Benzotriazole-5-carboxylic acid

Example 4 4-Nitro-1H-benzotriazole

Example 5 5-Amino-1H-benzotriazole

Example 6 5-Chloro-1H-benzotriazole

Example 7 5-Nitro-1H-benzotriazole

Example 8 4-[(1H-Benzotriazole-5-carbonyl)amino]benzoic acid

4-[(1H-Benzotriazole-5-carbonyl)amino]benzoic acid methyl ester (5.2 g,17.6 mmol) was dissolved in THF (60 mL) and methanol (10 mL) was addedfollowed by 1N sodium hydroxide (35 mL). The mixture was stirred at roomtemperature for 16 hours and then 1N hydrochloric acid (45 mL) wasadded. The mixture was added water (200 mL) and extracted with ethylacetate (2×500 mL). The combined organic phases were evaporated in vacuoto afford 0.44 g of 4-[(1H-benzotriazole-5-carbonyl)amino]benzoic acid.By filtration of the aqueous phase a further crop of4-[(1H-benzotriazole-5-carbonyl)amino]benzoic acid was isolated (0.52g).

¹H-NMR (DMSO-d₆): δ 7.97 (4H, s), 8.03 (2H, m), 8.66 (1H, bs), 10.7 (1H,s), 12.6 (1H, bs); HPLC-MS (Method A): m/z: 283 (M+1); Rt=1.85 min.

General Procedure (A) for Preparation of Compounds of General FormulaI₁:

wherein U, J and R²⁰ are as defined above, and J is optionallycontaining up to three substituents, R²², R²³ and R²⁴ as defined above.

The carboxylic acid of 1H-benzotriazole-5-carboxylic acid is activated,ie the OH functionality is converted into a leaving group L (selectedfrom eg fluorine, chlorine, bromine, iodine, 1-imidazolyl,1,2,4-triazolyl, 1-benzotriazolyloxy, 1-(4-aza benzotriazolyl)oxy,pentafluorophenoxy, N-succinyloxy3,4-dihydro-4-oxo-3-(1,2,3-benzotriazinyl)oxy, benzotriazole 5-COO, orany other leaving group known to act as a leaving group in acylationreactions. The activated benzotriazole-5-carboxylic acid is then reactedwith R²—(CH₂)_(n)—B′ in the presence of a base. The base can be eitherabsent (i.e. R²—(CH₂)_(n)—B′ acts as a base) or triethylamine,N-ethyl-N,N-diisopropylamine, N-methylmorpholine, 2,6-lutidine,2,2,6,6-tetramethylpiperidine, potassium carbonate, sodium carbonate,caesium carbonate or any other base known to be useful in acylationreactions. The reaction is performed in a solvent such as THF, dioxane,toluene, dichloromethane, DMF, NMP or a mixture of two or more of these.The reaction is performed between 0° C. and 80° C., preferably between20° C. and 40° C. When the acylation is complete, the product isisolated by extraction, filtration, chromatography or other methodsknown to those skilled in the art.

The general procedure (A) is further illustrated in the followingexample:

Example 9 General Procedure (A) 1H-Benzotriazole-5-carboxylic acidphenylamide

Benzotriazole-5-carboxylic acid (856 mg), HOAt (715 mg) and EDAC (1.00g) were dissolved in DMF (17.5 mL) and the mixture was stirred at roomtemperature 1 hour. A 0.5 mL aliquot of this mixture was added toaniline (13.7 μL, 0.15 mmol) and the resulting mixture was vigorouslyshaken at room temperature for 16 hours. 1N hydrochloric acid (2 mL) andethyl acetate (1 mL) were added and the mixture was vigorously shaken atroom temperature for 2 hours. The organic phase was isolated andconcentrated in vacuo to afford the title compound.

HPLC-MS (Method B): m/z: 239 (M+1); Rt=3.93 min.

The compounds in the following examples were similarly made. Optionally,the compounds may be isolated by filtration or by chromatography.

Example 10 General Procedure (A) 1H-Benzotriazole-5-carboxylic acid(4-methoxyphenyl)amide

HPLC-MS (Method A): m/z: 269 (M+1) & 291 (M+23); Rt=2.41 min

HPLC-MS (Method B): m/z: 239 (M+1); Rt=3.93 min.

Example 11 General Procedure (A){4-[(1H-Benzotriazole-5-carbonyl)amino]phenyl}carbamic acid tert-butylester

HPLC-MS (Method B): m/z: 354 (M+1); Rt=4.58 min.

Example 12 General Procedure (A) 1H-Benzotriazole-5-carboxylic acid(4-acetylaminophenyl)amide

HPLC-MS (Method B): m/z: 296 (M+1); Rt=3.32 min.

Example 13 General Procedure (A) 1H-Benzotriazole-5-carboxylic acid(3-fluorophenyl)amide

HPLC-MS (Method B): m/z: 257 (M+1); Rt=4.33 min.

Example 14 General Procedure (A) 1H-Benzotriazole-5-carboxylic acid(2-chlorophenyl)amide

HPLC-MS (Method B): m/z: 273 (M+1); Rt=4.18 min.

Example 15 General Procedure (A)4-[(1H-Benzotriazole-5-carbonyl)amino]benzoic acid methyl ester

HPLC-MS (Method A): m/z: 297 (M+1); Rt: 2.60 min. HPLC-MS (Method B):m/z: 297 (M+1); Rt=4.30 min.

Example 16 General Procedure (A) 1H-Benzotriazole-5-carboxylic acid(4-butylphenyl)amide

HPLC-MS (Method B): m/z: 295 (M+1); Rt=5.80 min.

Example 17 General Procedure (A) 1H-Benzotriazole-5-carboxylic acid(1-phenylethyl)amide

HPLC-MS (Method B): m/z: 267 (M+1); Rt=4.08 min.

Example 18 General Procedure (A) 1H-Benzotriazole-5-carboxylic acidbenzylamide

HPLC-MS (Method B): m/z: 253 (M+1); Rt=3.88 min.

Example 19 General Procedure (A) 1H-Benzotriazole-5-carboxylic acid4-chlorobenzylamide

HPLC-MS (Method B): m/z: 287 (M+1); Rt=4.40 min.

Example 20 General Procedure (A) 1H-Benzotriazole-5-carboxylic acid2-chlorobenzylamide

HPLC-MS (Method B): m/z: 287 (M+1); Rt=4.25 min.

Example 21 General Procedure (A) 1H-Benzotriazole-5-carboxylic acid4-methoxybenzylamide

HPLC-MS (Method B): m/z: 283 (M+1); Rt=3.93 min.

Example 22 General Procedure (A) 1H-Benzotriazole-5-carboxylic acid3-methoxybenzylamide

HPLC-MS (Method B): m/z: 283 (M+1); Rt=3.97 min.

Example 23 General Procedure (A) 1H-Benzotriazole-5-carboxylic acid(1,2-diphenylethyl)amide

HPLC-MS (Method B): m/z: 343 (M+1); Rt=5.05 min.

Example 24 General Procedure (A) 1H-Benzotriazole-5-carboxylic acid3-bromobenzylamide

HPLC-MS (Method B): m/z: 331 (M+1); Rt=4.45 min.

Example 25 General Procedure (A)4-{[(1H-Benzotriazole-5-carbonyl)amino]methyl}benzoic acid

HPLC-MS (Method B): m/z: 297 (M+1); Rt=3.35 min.

Example 26 General Procedure (A) 1H-Benzotriazole-5-carboxylic acidphenethylamide

HPLC-MS (Method B): m/z: 267 (M+1); Rt=4.08 min.

Example 27 General Procedure (A) 1H-Benzotriazole-5-carboxylic acid[2-(4-chlorophenyl)ethyl]amide

HPLC-MS (Method B): m/z: 301 (M+1); Rt=4.50 min.

Example 28 General Procedure (A) 1H-Benzotriazole-5-carboxylic acid[2-(4-methoxyphenyl)ethyl]amide

HPLC-MS (Method B): m/z: 297 (M+1); Rt=4.15 min.

Example 29 General Procedure (A) 1H-Benzotriazole-5-carboxylic acid[2-(3-methoxyphenyl)ethyl]amide

HPLC-MS (Method B): m/z: 297 (M+1); Rt=4.13 min.

Example 30 General Procedure (A) 1H-Benzotriazole-5-carboxylic acid[2-(3-chlorophenyl)ethyl]amide

HPLC-MS (Method B): m/z: 301 (M+1); Rt=4.55 min.

Example 31 General Procedure (A) 1H-Benzotriazole-5-carboxylic acid(2,2-diphenylethyl)amide

HPLC-MS (Method B): m/z: 343 (M+1); Rt=5.00 min.

Example 32 General Procedure (A) 1H-Benzotriazole-5-carboxylic acid(3,4-dichlorophenyl)methylamide

HPLC-MS (Method B): m/z: 321 (M+1); Rt=4.67 min.

Example 33 General Procedure (A) 1H-Benzotriazole-5-carboxylic acidmethylphenylamide

HPLC-MS (Method B): m/z: 253 (M+1); Rt=3.82 min.

Example 34 General Procedure (A) 1H-Benzotriazole-5-carboxylic acidbenzylmethylamide

HPLC-MS (Method B): m/z: 267 (M+1); Rt=4.05 min.

Example 35 General Procedure (A) 1H-Benzotriazole-5-carboxylic acid[2-(3-chloro-4-methoxyphenyl)ethyl]methyl-amide

HPLC-MS (Method B): m/z: 345 (M+1); Rt=4.37 min.

Example 36 General Procedure (A) 1H-Benzotriazole-5-carboxylic acidmethylphenethylamide

HPLC-MS (Method B): m/z 281 (M+1); Rt=4.15 min.

Example 37 General Procedure (A) 1H-Benzotriazole-5-carboxylic acid[2-(3,4-dimethoxyphenyl)ethyl]methylamide

HPLC-MS (Method B): m/z: 341 (M+1); Rt=3.78 min;

Example 38 General Procedure (A) 1H-Benzotriazole-5-carboxylic acid(2-hydroxy-2-phenylethyl)methylamide

HPLC-MS (Method B): m/z: 297 (M+1); Rt=3.48 min.

Example 39 General Procedure (A) 1H-Benzotriazole-5-carboxylic acid(3-bromophenyl)amide

HPLC-MS (Method A): m/z: 317 (M+1); Rt=3.19 min.

Example 40 General Procedure (A) 1H-Benzotriazole-5-carboxylic acid(4-bromophenyl)amide

HPLC-MS (Method A): m/z: 317 (M+1); Rt=3.18 min.

Example 41 General Procedure (A){4-[(1H-Benzotriazole-5-carbonyl)amino]benzoylamino}acetic acid

HPLC-MS (Method A): m/z: 340 (M+1); Rt=1.71 min.

Example 42 General Procedure (A){4-[(1H-Benzotriazole-5-carbonyl)amino]phenyl}acetic acid

HPLC-MS (Method A): m/z: 297 (M+1); Rt=2.02 min.

Example 43 General Procedure (A)3-{4-[(1H-Benzotriazole-5-carbonyl)amino]phenyl}acrylic acid

HPLC-MS (Method A): m/z: 309 (M+1); Rt=3.19 min.

Example 44 General Procedure (A){3-[(1H-Benzotriazole-5-carbonyl)amino]phenyl}acetic acid

HPLC-MS (Method A): m/z: 297 (M+1); Rt=2.10 min.

Example 45 General Procedure (A)2-{4-[(1H-Benzotriazole-5-carbonyl)amino]phenoxy}-2-methylpropionic acid

HPLC-MS (Method A): m/z: 341 (M+1); Rt=2.42 min.

Example 46 General Procedure (A)3-{4-[(1H-Benzotriazole-5-carbonyl)amino]benzoylamino}propionic acid

HPLC-MS (Method A): m/z: 354 (M+1); Rt=1.78 min.

Example 47 General Procedure (A)3-{4-[(1H-Benzotriazole-5-carbonyl)amino]phenyl}propionic acid

HPLC-MS (Method A): m/z: 311 (M+1); Rt=2.20 min.

Example 48 General Procedure (A) 1H-Benzotriazole-5-carboxylic acid(4-benzyloxyphenyl)amide

HPLC-MS (Method A): m/z: 345 (M+1); Rt=3.60 min.

Example 49 General Procedure (A) 1H-Benzotriazole-5-carboxylic acid(3-chloro-4-methoxyphenyl)amide

HPLC-MS (Method A): m/z: 303 (M+1); Rt=2.88 min.

Example 50 General Procedure (A) 1H-Benzotriazole-5-carboxylic acid(4-phenoxyphenyl)amide

HPLC-MS (Method A): m/z: 331 (M+1); Rt=3.62 min.

Example 51 General Procedure (A) 1H-Benzotriazole-5-carboxylic acid(4-butoxyphenyl)amide

HPLC-MS (Method A): m/z: 311 (M+1); Rt=3.59 min.

Example 52 General Procedure (A) 1H-Benzotriazole-5-carboxylic acid(3-bromo-4-trifluoromethoxyphenyl)amide

HPLC-MS (Method A): m/z: 402 (M+1); Rt=3.93 min.

Example 53 General Procedure (A) 1H-Benzotriazole-5-carboxylic acid(3,5-dichloro-4-hydroxyphenyl)amide

HPLC-MS (Method A): m/z: 323 (M+1); Rt=2.57 min.

Example 54 General Procedure (A)4-{[(1H-Benzotriazole-5-carbonyl)amino]methyl}benzoic acid

HPLC-MS (Method A): m/z: 297 (M+1); Rt=1.86 min.

Example 55 General Procedure (A){4-[(1H-Benzotriazole-5-carbonyl)amino]phenylsulfanyl}acetic acid

HPLC-MS (Method A): m/z: 329 (M+1); Rt=2.34 min.

Example 56 N-(1H-Benzotriazol-5-yl)acetamide

HPLC-MS (Method A): m/z: 177 (M+1); Rt=0.84 min.

Example 57 General Procedure (A) 1H-Benzotriazole-5-carboxylic acid4-nitrobenzylamide

General Procedure (B) for Preparation of Compounds of General FormulaI₂:

wherein X, Y, E and R¹⁰ are as defined above and E is optionallycontaining up to four optional substituents, R¹³, R¹⁴, R¹⁵, and R^(15A)as defined above.

The chemistry is well known (eg Lohray et al., J. Med. Chem., 1999, 42,2569-81) and is generally performed by reacting a carbonyl compound(aldehyde or ketone) with the heterocyclic ring (egthiazolidine-2,4-dione (X=O; Y=S), rhodanine (X=Y=S) and hydantoin (X=O;Y=NH) in the presence of a base, such as sodium acetate, potassiumacetate, ammonium acetate, piperidinium benzoate or an amine (egpiperidine, triethylamine and the like) in a solvent (eg acetic acid,ethanol, methanol, DMSO, DMF, NMP, toluene, benzene) or in a mixture oftwo or more of these solvents. The reaction is performed at roomtemperature or at elevated temperature, most often at or near theboiling point of the mixture. Optionally, azeotropic removal of theformed water can be done.

This general procedure (B) is further illustrated in the followingexample:

Example 58 General Procedure (B)5-(3-Phenoxybenzylidene)thiazolidine-2,4-dione

A solution of thiazolidine-2,4-dione (90%, 78 mg, 0.6 mmol) and ammoniumacetate (92 mg, 1.2 mmol) in acetic acid (1 mL) was added to3-phenoxybenzaldehyde (52 μL, 0.6 mmol) and the resulting mixture wasshaken at 115° C. for 16 hours. After cooling, the mixture wasconcentrated in vacuo to afford the title compound.

HPLC-MS (Method A): m/z: 298 (M+1); Rt=4.54 min.

The compounds in the following examples were similarly prepared.Optionally, the compounds can be further purified by filtration andwashing with water, ethanol and/or heptane instead of concentration invacuo. Also optionally the compounds can be purified by washing withethanol, water and/or heptane, or by chromatography, such as preparativeHPLC.

Example 59 General Procedure (B)5-(4-Dimethylaminobenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 249 (M+1); Rt=4.90 min

Example 60 General Procedure (B)5-Naphthalen-1-ylmethylenethiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 256 (M+1); Rt=4.16 min.

Example 61 General Procedure (B) 5-Benzylidene-thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 206 (M+1); Rt=4.87 min.

Example 62 General Procedure (B)5-(4-Methoxy-benzylidene)-thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 263 (M+1); Rt=4.90 min.

Example 63 General Procedure (B)5-(4-Chloro-benzylidene)-thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 240 (M+1); Rt=5.53 min.

Example 64 General Procedure (B)5-(4-Nitro-benzylidene)-thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 251 (M+1); Rt=4.87 min.

Example 65 General Procedure (B)5-(4-Hydroxy-3-methoxy-benzylidene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 252 (M+1); Rt=4.07 min.

Example 66 General Procedure (B)5-(4-Methylsulfanyl-benzylidene)-thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 252 (M+1); Rt=5.43 min.

Example 67 General Procedure (B)5-(3-Fluoro-4-methoxy-benzylidene)-thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 354 (M+1); Rt=4.97 min.

Example 68 General Procedure (B)5-(4-tert-Butylbenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 262 (M+1); Rt=6.70 min.

Example 69 General Procedure (B)N-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenyl]acetamide

HPLC-MS (Method A): m/z: 263 (M+1); Rt=3.90 min.

Example 70 General Procedure (B)5-Biphenyl-4-ylmethylene-thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 282 (M+1); Rt=4.52 min.

Example 71 General Procedure (B)5-(4-Phenoxy-benzylidene)-thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 298 (M+1); Rt=6.50 min.

Example 72 General Procedure (B)5-(3-Benzyloxybenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 312 (M+1); Rt=6.37 min.

Example 73 General Procedure (B)5-(3-p-Tolyloxybenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 312 (M+1); Rt=6.87 min.

Example 74 General Procedure (B)5-Napthalen-2-ylmethylene-thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 256 (M+1); Rt=4.15 min.

Example 75 General Procedure (B)5-Benzo[1,3]dioxol-5-ylmethylenethiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 250 (M+1), Rt=3.18 min.

Example 76 General Procedure (B)5-(4-Chlorobenzylidene)-2-thioxothiazolidin-4-one

HPLC-MS (Method A): m/z: 256 (M+1); Rt=4.51 min.

Example 77 General Procedure (B)5-(4-Dimethylaminobenzylidene)-2-thioxothiazolidin-4-one

HPLC-MS (Method A): m/z: 265 (M+1); Rt=5.66 min.

Example 78 General Procedure (B)5-(4-Nitrobenzylidene)-2-thioxothiazolidin-4-one

HPLC-MS (Method A): m/z: 267 (M+1); Rt=3.94 min.

Example 79 General Procedure (B)5-(4-Methylsulfanylbenzylidene)-2-thioxothiazolidin-4-one

HPLC-MS (Method A): m/z: 268 (M+1); Rt=6.39 min.

Example 80 General Procedure (B)5-(3-Fluoro-4-methoxybenzylidene)-2-thioxothiazolidin-4-one

HPLC-MS (Method A): m/z: 270 (M+1); Rt=5.52 min.

Example 81 General Procedure (B)5-Naphthalen-2-ylmethylene-2-thioxothiazolidin-4-one

HPLC-MS (Method A): m/z: 272 (M+1); Rt=6.75 min.

Example 82 General Procedure (B)5-(4-Diethylaminobenzylidene)-2-thioxothiazolidin-4-one

HPLC-MS (Method A): m/z: 293 (M+1); Rt=5.99 min.

Example 83 General Procedure (B)5-Biphenyl-4-ylmethylene-2-thioxothiazolidin-4-one

HPLC-MS (Method A): m/z: 298 (M+1); Rt=7.03 min.

Example 84 General Procedure (B)5-(3-Phenoxybenzylidene)-2-thioxothiazolidin-4-one

HPLC-MS (Method A): m/z: 314 (M+1); Rt=6.89 min.

Example 85 General Procedure (B)5-(3-Benzyloxybenzylidene)-2-thioxothiazolidin-4-one

HPLC-MS (Method A): m/z: 328 (M+1); Rt=6.95 min.

Example 86 General Procedure (B)5-(4-Benzyloxybenzylidene)-2-thioxothiazolidin-4-one

HPLC-MS (Method A): m/z: 328 (M+1); RT=6.89 min.

Example 87 General Procedure (B)5-Naphthalen-1-ylmethylene-2-thioxothiazolidin-4-one

HPLC-MS (Method A): m/z: 272 (M+1); Rt=6.43 min.

Example 88 General Procedure (B)5-(3-Methoxybenzyl)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 236 (M+1); Rt=3.05 min.

Example 89 General Procedure (D)4-[2-Chloro-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyric acidethyl ester

HPLC-MS (Method A): m/z: 392 (M+23), Rt=4.32 min.

Example 90 General Procedure (D)4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)-phenoxy]-butyricacid

HPLC-MS (Method A): m/z: 410 (M+23); Rt=3.35 min.

Example 91 General Procedure (B)5-(3-Bromobenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 285 (M+1); Rt=4.01 min.

Example 92 General Procedure (B)5-(4-Bromobenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 285 (M+1); Rt=4.05 min.

Example 93 General Procedure (B)5-(3-Chlorobenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 240 (M+1); Rt=3.91 min.

Example 94 General Procedure (B)5-Thiophen-2-ylmethylenethiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 212 (M+1); Rt=3.09 min.

Example 95 General Procedure (B)5-(4-Bromothiophen-2-ylmethylene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 291 (M+1); Rt=3.85 min.

Example 96 General Procedure (B)5-(3,5-Dichlorobenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 274 (M+1); Rt=4.52 min.

Example 97 General Procedure (B)5-(1-Methyl-1H-indol-3-ylmethylene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 259 (M+1); Rt=3.55 min.

Example 98 General Procedure (B)5-(1H-Indol-3-ylmethylene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 245 (M+1); Rt=2.73 min.

Example 99 General Procedure (B)5-Fluoren-9-ylidenethiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 280 (M+1); Rt=4.34 min.

Example 100 General Procedure (B)5-(1-Phenylethylidene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 220 (M+1); Rt=3.38 min.

Example 101 General Procedure (B)5-[1-(4-Methoxyphenyl)-ethylidene]-thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 250 (M+1); Rt=3.55 min.

Example 102 General Procedure (B)5-(1-Naphthalen-2-yl-ethylidene)-thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 270 (M+1); Rt=4.30 min.

Example 103 General Procedure (B)5-[1-(4-Bromophenyl)-ethylidene]-thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 300 (M+1); Rt=4.18 min.

Example 104 General Procedure (B)5-(2,2-Diphenylethylidene)-thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 296 (M+1); Rt=4.49 min.

Example 105 General Procedure (B)5-[1-(3-Methoxyphenyl)-ethylidene]-thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 250 (M+1); Rt=3.60 min.

Example 106 General Procedure (B)5-[1-(6-Methoxynaphthalen-2-yl)-ethylidene]-thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 300 (M+1); Rt=4.26 min.

Example 107 General Procedure (B)5-[1-(4-Phenoxyphenyl)-ethylidene]-thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 312 (M+1); Rt=4.68 min.

Example 108 General Procedure (B)5-[1-(3-Fluoro-4-methoxyphenyl)ethylidene]thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 268 (M+1); Rt=3.58 min.

Example 109 General Procedure (B)5-[1-(3-Bromophenyl)-ethylidene]-thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 300 (M+1); Rt=4.13 min.

Example 110 General Procedure (B)5-Anthracen-9-ylmethylenethiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 306 (M+1); Rt=4.64 min.

Example 111 General Procedure (B)5-(2-Methoxynaphthalen-1-ylmethylene)-thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 286 (M+1); Rt=4.02 min.

Example 112 General Procedure (B)5-(4-Methoxynaphthalen-1-ylmethylene)-thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 286 (M+1); Rt=4.31 min.

Example 113 General Procedure (B)5-(4-Dimethylaminonaphthalen-1-ylmethylene)-thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 299 (M+1); Rt=4.22 min.

Example 114 General Procedure (B)5-(4-Methylnaphthalen-1-ylmethylene)-thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 270 (M+1); Rt=4.47 min.

Example 115 General Procedure (B)5-Pyridin-2-ylmethylene-thiazolidine-2,4-dione

Example 116 5-Pyridin-2-ylmethyl-thiazolidine-2,4-dione

5-Pyridin-2-ylmethylene-thiazolidine-2,4-dione (5 g) in tetrahydrofuran(300 ml) was added 10% Pd/C (1 g) and the mixture was hydrogenated atambient pressure for 16 hours. More 10% Pd/C (5 g) was added and themixture was hydrogenated at 50 psi for 16 hours. After filtration andevaporation in vacuo, the residue was purified by column chromatographyeluting with a mixture of ethyl acetate and heptane (1:1). This affordedthe title compound (0.8 g, 16%) as a solid.

TLC: R_(f)=0.30 (SiO₂; EtOAc:heptane 1:1)

Example 117 General Procedure (B)5-(1H-Imidazol-4-ylmethylene)-thiazolidine-2,4-dione

Example 118 General Procedure (B)5-(4-Benzyloxy-benzylidene)-thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 6.43 min; 99% (2A)

Example 119 General Procedure (B)5-[4-(4-Fluorobenzyloxy)benzylidene]-2-thioxothiazolidin-4-one

Example 120 General Procedure (B)5-(4-Butoxybenzylidene)-2-thioxothiazolidin-4-one

Example 121 General Procedure (B)5-(3-Methoxybenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 236 (M+1); Rt=4.97 min

Example 122 General Procedure (B)5-(3-Methoxybenzylidene)imidazolidine-2,4-dione

HPLC-MS (Method A): m/z: 219 (M+1); Rt=2.43 min.

Example 123 General Procedure (B)5-(4-Methoxybenzylidene)imidazolidine-2,4-dione

HPLC-MS (Method A): m/z: 219 (M+1); Rt=2.38 min.

Example 124 General Procedure (B)5-(2,3-Dichlorobenzylidene)thiazolidine-2,4-dione

Example 125 General Procedure (B)5-Benzofuran-7-ylmethylenethiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 247 (M+1); Rt=4.57 min.

Example 126 General Procedure (B)5-Benzo[1,3]dioxol-4-ylmethylenethiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 250 (M+1); Rt=4.00 min.

Example 127 General Procedure (B)5-(4-Methoxy-2,3-dimethylbenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 264 (M+1); Rt=5.05 min.

Example 128 General Procedure (B)5-(2-Benzyloxy-3-methoxybenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 342 (M+1); Rt=5.14 min.

Example 129 General Procedure (B)5-(2-Hydroxybenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 222 (M+1); Rt=3.67 min.

Example 130 General Procedure (B)5-(2,4-Dichlorobenzylidene)thiazolidine-2,4-dione

¹H-NMR (DMSO-d₆): 7.60 (2H, “s”), 7.78 (1H, s), 7.82 (1H, s).

Example 131 General Procedure (B)5-(2-Chlorobenzylidene)thiazolidine-2,4-dione

¹H-NMR (DMSO-d₆): 7.40 (1H, t), 7.46 (1H, t), 7.57 (1H, d), 7.62 (1H,d), 7.74 (1H, s).

Example 132 General Procedure (B)5-(2-Bromobenzylidene)thiazolidine-2,4-dione

¹H-NMR (DMSO-d₆): 7.33 (1H, t), 7.52 (1H, t), 7.60 (1H, d), 7.71 (1H,s), 7.77 (1H, d).

Example 133 General Procedure (B)5-(2,4-Dimethoxybenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 266 (M+1) Rt=4.40 min.

Example 134 General Procedure (B)5-(2-Methoxybenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 236 (M+1); Rt=4.17 min.

Example 135 General Procedure (B)5-(2,6-Difluorobenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 242 (M+1); Rt=4.30 min.

Example 136 General Procedure (B)5-(2,4-Dimethylbenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 234 (M+1); Rt=5.00 min.

Example 137 General Procedure (B)5-(2,4,6-Trimethoxybenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 296 (M+1); Rt=4.27 min.

Example 138 General Procedure (B)5-(4-Hydroxy-2-methoxybenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 252 (M+1); Rt=3.64 min.

Example 139 General Procedure (B)5-(4-Hydroxynaphthalen-1-ylmethylene)thiazolidine-2,4-dione

¹H-NMR (DMSO-d₆): δ=7.04 (1H, d), 7.57 (2H, m), 7.67 (1H, t), 8.11 (1H,d), 8.25 (1H, d), 8.39 (1H, s) 11.1 (1H, s), 12.5 (1H, bs). HPLC-MS(Method C): m/z: 272 (M+1); Rt=3.44 min.

Example 140 General Procedure (B)5-(2-Trifluoromethoxybenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 290 (M+1); Rt=4.94 min.

Example 141 General Procedure (B)5-Biphenyl-2-ylmethylenethiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 282 (M+1); Rt=5.17 min.

Example 142 General Procedure (B)5-(2-Benzyloxybenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 312 (M+1); Rt=5.40 min.

Example 143 General Procedure (B)5-Adamantan-2-ylidenethiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 250 (M+1); Rt=4.30 min.

General Procedure (C) for Preparation of Compounds of General FormulaI₂:

wherein X, Y, E, and R¹⁰ are as defined above and E is optionallycontaining up to four optional substituents, R¹³, R¹⁴, R¹⁵, and R^(15A)as defined above.

This general procedure (C) is quite similar to general procedure (B) andis further illustrated in the following example:

Example 144 General Procedure (C)5-(3,4-Dibromobenzylidene)thiazolidine-2,4-dione

A mixture of thiazolidine-2,4-dione (90%, 65 mg, 0.5 mmol),3,4-dibromobenzaldehyde (132 mg, 0.5 mmol), and piperidine (247 μL, 2.5mmol) was shaken in acetic acid (2 mL) at 110° C. for 16 hours. Aftercooling, the mixture was concentrated to dryness in vacuo. The resultingcrude product was shaken with water, centrifuged, and the supernatantwas discarded. Subsequently the residue was shaken with ethanol,centrifuged, the supernatant was discarded and the residue was furtherevaporated to dryness to afford the title compound.

¹H NMR (Acetone-d₆): δ_(H) 7.99 (d, 1H), 7.90 (d, 1H), 7.70 (s, 1H),7.54 (d, 1H); HPLC-MS (Method A): m/z: 364 (M+1); Rt=4.31 min.

The compounds in the following examples were similarly prepared.Optionally, the compounds can be further purified by filtration andwashing with water instead of concentration in vacuo. Also optionallythe compounds can be purified by washing with ethanol, water and/orheptane, or by preparative HPLC.

Example 145 General Procedure (C)5-(4-Hydroxy-3-iodo-5-methoxybenzylidene)thiazolidine-2,4-dione

Mp=256° C.; ¹H NMR (DMSO-d₆) δ=12.5 (s, broad, 1H), 10.5 (s, broad, 1H),7.69 (s, 1H), 7.51 (d, 1H), 7.19 (d, 1H) 3.88 (s, 3H), ¹³C-NMR (DMSO-d₆)δ=168.0, 167.7, 149.0, 147.4, 133.0, 131.2, 126.7, 121.2, 113.5, 85.5,56.5; HPLC-MS (Method A): m/z: 378 (M+1); Rt=3.21 min.

Example 146 General Procedure (C)5-(4-Hydroxy-2,6-dimethylbenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method C): m/z 250 (M+1); Rt.=2.45 min.

Example 147 General Procedure (C)4-[5-Bromo-6-(2,4-dioxothiazolidin-5-ylidenemethyl)-naphthalen-2-yloxymethyl]-benzoicacid

HPLC-MS (Method C): m/z: 506 (M+23); Rt.=4.27 min.

Example 148 General Procedure (C)5-(4-Bromo-2,6-dichlorobenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 354 (M+1); Rt.=4.36 min.

Example 149 General Procedure (C)5-(6-Hydroxy-2-naphthylmethylene)thiazolidine-2,4-dione

Mp 310-314° C., ¹H NMR (DMSO-d₆): δ_(H)=12.5 (s, broad, 1H), 8.06 (d,1H), 7.90-7.78 (m, 2H), 7.86 (s, 1H), 7.58 (dd, 1H), 7.20 7.12 (m, 2H).¹³C NMR (DMSO-d₆): δ_(C)=166.2, 165.8, 155.4, 133.3, 130.1, 129.1,128.6, 125.4, 125.3, 125.1, 124.3, 120.0, 117.8, 106.8; HPLC-MS (MethodA): m/z: 272 (M+1); Rt=3.12 min.

Preparation of the Starting Material, 6-hydroxy-2-naphtalenecarbaldehyde

6-Cyano-2-naphthalenecarbaldehyde (1.0 g, 5.9 mmol) was dissolved in dryhexane (15 mL) under nitrogen. The solution was cooled to −60° C. and asolution of diisobutyl aluminium hydride (DIBAH) (15 mL, 1M in hexane)was added dropwise. After the addition, the solution was left at roomtemperature overnight. Saturated ammonium chloride solution (20 mL) wasadded and the mixture was stirred at room temperature for 20 min,subsequently aqueous H₂SO₄ (10% solution, 15 mL) was added followed bywater until all salt was dissolved. The resulting solution was extractedwith ethyl acetate (3×), the combined organic phases were dried withMgSO₄, evaporated to dryness to afford 0.89 g of6-hydroxy-2-naphtalenecarbaldehyde.

Mp.: 153.5-156.5° C.; HPLC-MS (Method A): m/z: 173 (M+1); Rt=2.67 min;¹H NMR (DMSO-d₆): δ_(H)=10.32 (s, 1H), 8.95 (d, 1H), 10.02 (s, 1H), 8.42(s, broad, 1H), 8.01 (d, 1H), 7.82-7.78 (m, 2H), 7.23-7.18 (m, 2H).

Alternative Preparation of 6-hydroxy-2-naphtalenecarbaldehyde

To a stirred cooled mixture of 6-bromo-2-hydroxynaphthalene (25.3 g,0.113 mol) in THF (600 mL) at −78° C. was added n-BuLi (2.5 M, 100 mL,0.250 mol) dropwise. The mixture turned yellow and the temperature roseto −64° C. After ca 5 min a suspension appeared. After addition, themixture was maintained at −78° C. After 20 minutes, a solution of DMF(28.9 mL, 0.373 mol) in THF (100 mL) was added over 20 minutes. Afteraddition, the mixture was allowed to warm slowly to RT. After 1 hour,the mixture was poured in ice/water (200 mL). To the mixture citric acidwas added to a pH of 5. The mixture was stirred for 0.5 hour. Ethylacetate (200 mL) was added and the organic layer was separated andwashed with brine (100 mL), dried over Na₂SO₄ and concentrated. To theresidue was added heptane with 20% ethyl acetate (ca 50 mL) and themixture was stirred for 1 hour. The mixture was filtered and the solidwas washed with ethyl acetate and dried in vacuo to afford 16 g of thetitle compound.

Example 150 General Procedure (C)5-(3-Iodo-4-methoxybenzylidene)thiazolidiene-2,4-dione

¹H NMR (DMSO-d₆): δ_(H) 12.55 (s, broad, 1H), 8.02 (d, 1H), 7.72 (s, 1H)7.61 (d, 1H), 7.18 (d, 1H), 3.88 (s, 3H); ¹³C NMR (DMSO-d₆); δ_(C)168.1, 167.7, 159.8, 141.5, 132.0, 130.8, 128.0, 122.1, 112.5, 87.5,57.3. HPLC-MS (Method A): m/z: 362 (M+1); Rt=4.08 min.

Preparation of the Starting Material, 3-iodo-4-methoxybenzaldehyde

4-Methoxybenzaldehyde (0.5 g, 3.67 mmol) and silver trifluoroacetate(0.92 g, 4.19 mmol) were mixed in dichloromethane (25 mL). Iodine (1.19g, 4.7 mmol) was added in small portions and the mixture was stirredovernight at room temperature under nitrogen. The mixture wassubsequently filtered and the residue washed with DCM. The combinedfiltrates were treated with an aqueous sodium thiosulfate solution (1 M)until the colour disappeared. Subsequent extraction with dichloromethane(3×20 mL) followed by drying with MgSO₄ and evaporation in vacuoafforded 0.94 g of 3-iodo-4-methoxybenzaldehyde.

Mp 104-107° C.; HPLC-MS (Method A): m/z: 263 (M+1); Rt=3.56 min.; ¹H NMR(CDCl₃): δ_(H)=8.80 (s, 1H), 8.31 (d, 1H), 7.85 (dd, 1H) 6.92 (d, 1H),3.99 (s, 3H).

Example 151 General Procedure (C)5-(1-Bromonaphthalen-2-ylmethylene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z:=336 (M+1); Rt=4.46 min.

Example 152 General Procedure (C)1-[5-(2,4-Dioxothiazolidin-5-ylidenemethyl)thiazol-2-yl]piperidine-4-carboxylicacid ethyl ester

¹H NMR (DMSO-d₆): δ_(H)=7.88 (s, 1H), 7.78 (s, 1H), 4.10 (q, 2H),4.0-3.8 (m, 2H), 3.40-3.18 (m, 2H), 2.75-2.60 (m, 1H), 2.04-1.88 (m,2H), 1.73-1.49 (m, 2H), 1.08 (t, 3H); HPLC-MS (Method A): m/z: 368(M+1); Rt=3.41 min.

Example 153 General Procedure (C)5-(2-Phenyl-[1,2,3]triazol-4-ylmethylene)thiazolidine-2,4-dione

¹H NMR (DMSO-d₆): δ_(H)=12.6 (s, broad, 1H), 8.46 (s, 1H), 8.08 (dd,2H), 7.82 (s, 1H), 7.70-7.45 (m, 3H). HPLC-MS (Method A): m/z: 273(M+1); Rt=3.76 min.

Example 154 General Procedure (C)5-(Quinolin-4-ylmethylene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 257 (M+1); Rt=2.40 min.

Example 155 General Procedure (C)5-(6-Methylpyridin-2-ylmethylene)thiazolidine-2,4-dione

¹H NMR (DMSO-d₆): δ_(H)=12.35 (s, broad, 1H), 7.82 (t, 1H), 778 (s, 1H),7.65 (d, 1H), 7.18 (d, 1H), 2.52 (s, 3H); HPLC-MS (Method A): m/z: 221(M+1); Rt=3.03 min.

Example 156 General Procedure (C)5-(2,4-dioxothiazolidin-5-ylidenemethyl)-furan-2-ylmethylacetate

¹H NMR (DMSO-d₆): δ_(H)=12.46 (s, broad, 1H), 7.58 (s, 1H), 7.05 (d,1H), 6.74 (s, 1H), 5.13 (s, 2H), 2.10 (s, 3H). HPLC-MS (Method A): m/z:208 (M−CH₃COO); Rt=2.67 min.

Example 157 General Procedure (C)5-(2,4-Dioxothiazolidin-5-ylidenemethyl)furan-2-sulfonic acid

HPLC-MS (Method A): m/z: 276 (M+1); Rt=0.98 min.

Example 158 General Procedure (C)5-(5-Benzyloxy-1H-pyrrolo[2,3-c]pyridin-3-ylmethylene)-thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 352 (M+1); Rt=3.01 min.

Example 159 General Procedure (C)5-(Quinolin-2-ylmethylene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 257 (M+1); Rt=3.40 min.

Example 160 General Procedure (C)5-(2,4-Dioxothiazolidin-5-ylidenemethyl)thiophene-2-carboxylic acid

HPLC-MS (Method A): m/z: 256 (M+1); Rt=1.96 min.

Example 161 General Procedure (C)5-(2-Phenyl-1H-imidazol-4-ylmethylene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 272 (M+1); Rt=2.89 min.

Example 162 General Procedure (C)5-(4-Imidazol-1-yl-benzylidene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 272 (M+1); Rt=1.38 min.

Example 163 General Procedure (C)5-(9-Ethyl-9H-carbazol-3-ylmethylene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 323 (M+1); Rt=4.52 min.

Example 164 General Procedure (C)5-(1,4-Dimethyl-9H-carbazol-3-ylmethylene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 323 (M+1); Rt=4.35 min.

Example 165 General Procedure (C)5-(2-Methyl-1H-indol-3-ylmethylene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 259 (M+1); Rt=3.24 min.

Example 166 General Procedure (C)5-(2-Ethylindol-3-ylmethylene)thiazolidine-2,4-dione

2-Methylindole (1.0 g, 7.6 mmol) dissolved in diethyl ether (100 mL)under nitrogen was treated with n-Butyl lithium (2 M in pentane, 22.8mmol) and potassium tert-butoxide (15.2 mmol) with stirring at RT for 30min. The temperature was lowered to −70 C. and methyl Iodide (15.2 mmol)was added and the resulting mixture was stirred at −70 for 2 h. Then 5drops of water was added and the mixture allowed to warm up to RT.Subsequently, the mixture was poured into water (300 mL), pH wasadjusted to 6 by means of 1N hydrochloric acid and the mixture wasextracted with diethyl ether. The organic phase was dried with Na₂SO₄and evaporated to dryness. The residue was purified by columnchromatography on silica gel using heptane/ether (4/1) as eluent. Thisafforded 720 mg (69%) of 2-ethylindole.

¹H NMR (DMSO-d₆): δ=10.85 (1H, s); 7.39 (1H, d); 7.25 (1H, d); 6.98 (1H,t); 6.90 (1H, t); 6.10 (1H, s); 2.71 (2H, q); 1.28 (3H, t).

2-Ethylindole (0.5 g, 3.4 mmol) dissolved in DMF (2 mL) was added to acold (0° C.) premixed (30 minutes) mixture of DMF (1.15 mL) andphosphorous oxychloride (0.64 g, 4.16 mmol). After addition of2-ethylindole, the mixture was heated to 40° C. for 1 h, water (5 mL)was added and the pH adjusted to 5 by means of 1 N sodium hydroxide. Themixture was subsequently extracted with diethyl ether, the organic phaseisolated, dried with MgSO₄ and evaporated to dryness affording2-ethylindole-3-carbaldehyde (300 mg).

HPLC-MS (Method C): m/z: 174 (M+1); Rt.=2.47 min.

2-Ethylindole-3-carbaldehyde (170 mg) was treated withthiazolidine-2,4-dione using the general procedure (C) to afford thetitle compound (50 mg).

HPLC-MS (Method C): m/z: 273 (M+1); Rt.=3.26 min.

Example 167 General Procedure (C)5-[2-(4-Bromophenylsulfanyl)-1-methyl-1H-indol-3-ylmethylene]thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 447 (M+1); Rt=5.25 min.

Example 168 General Procedure (C)5-[2-(2,4-Dichlorobenzyloxy)-naphthalen-1-ylmethylene]thiazolidine-2,4-dione

HPLC-MS (Method A): (anyone 1) m/z: 430 (M+1); Rt=5.47 min.

Example 169 General Procedure (C)5-{4-[3-(4-Bromophenyl)-3-oxopropenyl]-benzylidene}thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 416 (M+1); Rt=5.02 min.

Example 170 General Procedure (C)5-(4-Pyridin-2-ylbenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 283 (M+1), Rt=2.97 min.

Example 171 General Procedure (C)5-(3,4-Bisbenzyloxybenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 418 (M+1); Rt=5.13 min.

Example 172 General Procedure (C)5-[4-(4-Nitrobenzyloxy)-benzylidene]thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 357 (M+1); Rt=4.45 min.

Example 173 General Procedure (C)5-(2-Phenyl-1H-indol-3-ylmethylene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 321 (M+1); Rt=3.93 min.

Example 174 General Procedure (C)5-(5-Benzyloxy-1H-indol-3-ylmethylene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 351 (M+1); Rt=4.18 min.

Example 175 General Procedure (C)5-(4-Hydroxybenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method A); m/z: 222 (M+1); Rt=2.42 min.

Example 176 General Procedure (C)5-(1-Methyl-1H-indol-2-ylmethylene)thiazolidine-2,4-dione

¹H NMR (DMSO-d₆): δ_(H)=12.60 (s, broad, 1H), 7.85 (s, 1H), 7.68 (dd,1H), 7.55 (dd, 1H), 7.38 (dt, 1H), 7.11 (dt, 1H) 6.84 (s, 1H), 3.88 (s,3H); HPLC-MS (Method A): m/z: 259 (M+1); Rt=4.00 min.

Example 177 General Procedure (C)5-(5-Nitro-1H-indol-3-ylmethylene)thiazolidine-2,4-dione

Mp 330-333° C., ¹H NMR (DMSO-d₆): δ_(H)=12.62 (s, broad, 1H), 8.95 (d,1H), 8.20 (s, 1H), 8.12 (dd, 1H), 7.98 (s, broad, 1H), 7.68 (d, 1H);HPLC-MS (Method A): m/z: 290 (M+1); Rt=3.18 min.

Example 178 General Procedure (C)5-(6-Methoxynaphthalen-2-ylmethylene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 286 (M+1); Rt=4.27 min.

Example 179 General Procedure (C)5-(3-Bromo-4-methoxybenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 314 (M+1), Rt=3.96 min.

Example 180 General Procedure (C)3-{(2-Cyanoethyl)-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenyl]amino}propionitrile

HPLC-MS (Method A): m/z: 327 (M+1); Rt=2.90 min.

Example 181 General Procedure (C)3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indole-6-carboxylic acid methylester

HPLC-MS (Method A): m/z: 303 (M+1); Rt=3.22-3.90 min.

Example 182 3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indole-6-carboxylicacid pentyl ester

(3-(2,4-Dioxohiazolidin-5-ylidenemethyl)indole-6-carboxylic acid methylester (example 181, 59 mg; 0.195 mmol) was stirred in pentanol (20 mL)at 145° C. for 16 hours. The mixture was evaporated to dryness affordingthe title compound (69 mg).

HPLC-MS (Method C): m/z: 359 (M+1); Rt.=4.25 min.

Example 183 General Procedure (C)3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indole-7-carboxylic acid

HPLC-MS (Method A): m/z: 289 (M+1); Rt=2.67 min.

Example 184 General Procedure (C)5-(1-Benzylindol-3-ylmethylene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 335 (M+1); Rt=4.55 min.

Example 185 General Procedure (C)5-(1-Benzenesulfonylindol-3-ylmethylene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z:=385 (M+1); Rt=4.59 min.

Example 186 General Procedure (C)5-(4-[1,2,3]Thiadiazol-4-ylbenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 290 (M+1); Rt=3.45 min.

Example 187 General Procedure (C)5-[4-(4-Nitrobenzyloxy)-benzylidene]thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 357 (M+1); Rt=4.42 min.

Example 188 General Procedure (C)3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indole-1-carboxylic acid ethylester

HPLC-MS (Method A): m/z: 317 (M+1); Rt=4.35 min.

Example 189 General Procedure (C)5-[2-(4-Pentylbenzoyl)-benzofuran-5-ylmethylene]thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 420 (M+1); Rt=5.92 min.

Example 190 General Procedure (C)5-[1-(2-Fluorobenzyl)-4-nitroindol-3-ylmethylene]thiazolidine-2,4-dione

HPLC-MS (Method A): (Anyone 1) m/z: 398 (M+1); Rt=4.42 min.

Example 191 General Procedure (C)5-(4-Benzyloxyindol-3-ylmethylene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 351 (M+1); Rt=3.95 min.

Example 192 General Procedure (C)5-(4-Isobutylbenzylidene)-thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 262 (M+1); Rt=4.97 min.

Example 193 General Procedure (C) Trifluoromethanesulfonic acid4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yl ester

HPLC-MS (Method A): m/z: 404 (M+1); Rt=4.96 min.

Preparation of Starting Material:

4-Hydroxy-1-naphthaldehyde (10 g, 58 mmol) was dissolved in pyridin (50ml) and the mixture was cooled to 0-5° C. With stirring,trifluoromethanesulfonic acid anhydride (11.7 ml, 70 mmol) was addeddrop-wise. After addition was complete, the mixture was allowed to warmup to room temperature, and diethyl ether (200 ml) was added. Themixture was washed with water (2×250 ml), hydrochloric acid (3N, 200ml), and saturated aqueous sodium chloride (100 ml). After drying(MgSO4), filtration and concentration in vacuo, the residue was purifiedby column chromatography on silica gel eluting with a mixture of ethylacetate and heptane (1:4). This afforded 8.35 g (47%)trifluoromethanesulfonic acid 4-formylnaphthalen-1-yl ester, mp 44-46.6°C.

Example 194 General Procedure (C)5-(4-Nitroindol-3-ylmethylene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 290 (M+1); Rt=3.14 min.

Example 195 General Procedure (C)5-(3,5-Dibromo-4-hydroxy-benzylidene)thiazolidine-2,4-dione

¹H NMR (DMSO-d₆): δ_(H)=12.65 (broad, 1H), 10.85 (broad, 1H), 7.78 (s,2H), 7.70 (s, 1H);

HPLC-MS (Method A): m/z: 380 (M+1); Rt=3.56 min.

Example 196 General Procedure (C)

HPLC-MS (Method A): m/z: 385 (M+1); Rt=5.08 min.

General Procedure for Preparation of Starting Materials for Examples196-199:

Indole-3-carbaldehyde (3.8 g, 26 mmol) was stirred with potassiumhydroxide (1.7 g) in acetone (200 mL) at RT until a solution wasobtained indicating full conversion to the indole potassium salt.Subsequently the solution was evaporated to dryness in vacuo. Theresidue was dissolved in acetone to give a solution containing 2.6mmol/20 mL.

20 mL portions of this solution were mixed with equimolar amounts ofarylmethylbromides in acetone (10 mL). The mixtures were stirred at RTfor 4 days and subsequently evaporated to dryness and checked byHPLC-MS. The crude products, 1-benzylated indole-3-carbaldehydes, wereused for the reaction with thiazolidine-2,4-dione using the generalprocedure C.

Example 197 General Procedure (C)4-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indol-1-ylmethyl]benzoic acidmethyl ester

HPLC-MS (Method A): m/z: 393 (M+1); Rt=4.60 min.

Example 198 General Procedure (C)5-[1-(9,10-Dioxo-9,10-dihydroanthracen-2-ylmethyl)-1H-indol-3-ylmethylene]thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 465 (M+1); Rt=5.02 min.

Example 199 General Procedure (C)4′-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indol-1-ylmethyl]biphenyl-2-carbonitrile

HPLC-MS (Method A): m/z: 458 (M+23); Rt=4.81 min.

Example 200 General Procedure (C)3-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)-2-methylindol-1-ylmethyl]benzonitrile

2-Methylindole-3-carbaldehyde (200 mg, 1.26 mmol) was added to a slurryof 3-bromomethylbenzenecarbonitrile (1.26 mmol) followed by sodiumhydride, 60%, (1.26 mmol) in DMF (2 mL). The mixture was shaken for 16hours, evaporated to dryness and washed with water and ethanol. Theresidue was treated with thiazolidine-2,4-dione following the generalprocedure C to afford the title compound (100 mg).

HPLC-MS (Method C): m/z: 374 (M+1); Rt.=3.95 min.

Example 201 General Procedure (C)5-(1-Benzyl-2-methylindol-3-ylmethylene)thiazolidine-2,4-dione

This compound was prepared in analogy with the compound described inexample 200 from benzyl bromide and 2-methylindole-3-carbaldehyde,followed by reaction with thiazolidine-2,4-dione resulting in 50 mg ofthe title compound.

HPLC-MS (Method C): m/z: 349 (M+1); Rt.=4.19 min.

Example 2024-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)-2-methylindol-1-ylmethyl]benzoicacid methyl ester

This compound was prepared in analogy with the compound described inexample 200 from 4-(bromomethyl)benzoic acid methyl ester and2-methylindole-3-carbaldehyde, followed by reaction withthiazolidine-2,4-dione.

HPLC-MS (Method C): m/z: 407 (M+1); Rt.=4.19 min.

Example 203 General Procedure (C)5-(2-Chloro-1-methyl-1H-indol-3-ylmethylene)thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 293 (M+1); Rt=4.10 min.

Example 204 General Procedure (C)5-(4-Hydroxy-3,5-diiodo-benzylidene)-thiazolidine-2,4-dione

HPLC-MS (Method A): m/z: 474 (M+1); Rt=6.61 min.

Example 205 General Procedure (C)5-(4-Hydroxy-3-iodobenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 348 (M+1); Rt.=3.13 min ¹H-NMR): (DMSO-d₆):11.5 (1H, broad); 7.95 (1H, d); 7.65 (1H, s); 7.45 (1H, dd); 7.01 (1H,dd); 3.4 (1H, broad).

Example 206 General Procedure (C)5-(2,3,6-Trichlorobenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 309 (M+1); Rt.=4.07 min

Example 207 General Procedure (C)5-(2,6-Dichlorobenzylidene)thiazolidine-2,4-dione

Mp. 152-154° C.

HPLC-MS (Method C): m/z: 274 (M+1), Rt.=3.70 min

¹H-NMR: (DMSO-d₆): 12.8 (1H, broad); 7.72 (1H, s); 7.60 (2H, d); 7.50(1H, t).

Example 208 General Procedure (C)5-[1-(2,6-Dichloro-4-trifluoromethylphenyl)-2,5-dimethyl-1H-pyrrol-3-ylmethylene]thiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 436 (M+1); Rt.=4.81 min

Example 209 General Procedure (C)5-[1-(3,5-Dichlorophenyl)-5-(4-methanesulfonylphenyl)-2-methyl-1H-pyrrol-3-ylmethylene]-thiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 508 (M+1); Rt.=4.31 min

Example 210 General Procedure (C)5-[1-(2,5-Dimethoxyphenyl)-5-(4-methanesulfonylphenyl)-2-methyl-1H-pyrrol-3-ylmethylene]-thiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 499 (M+1); Rt.=3.70 min

Example 211 General Procedure (C)4-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)-2,5-dimethylpyrrol-1-yl]benzoicacid

HPLC-MS (Method C): m/z: 342 (M+1); Rt.=3.19 min

Example 212 General Procedure (C)5-(4-Hydroxy-2,6-dimethoxybenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 282 (M+1); Rt.=2.56, mp=331-333° C.

Example 213 General Procedure (C)5-(2,6-Dimethylbenzylidene)thiazolidine-2,4-dione

M.p: 104-105° C.

HPLC-MS (Method C): m/z: 234 (M+1); Rt.=3.58 min,

Example 214 General Procedure (C)5-(2,6-Dimethoxybenzylidene)thiazolidine-2,4-dione

Mp: 241-242° C.

HPLC-MS (Method C): m/z: 266 (M+1); Rt.=3.25 min;

Example 215 General Procedure (C)5-[4-(2-Fluoro-6-nitrobenzyloxy)-2,6-dimethoxybenzylidene]thiazolidine-2,4-dione

Mp: 255-256° C.

HPLC-MS (Method C): m/z: 435 (M+1), Rt=4.13 min,

Example 216 General Procedure (C)5-Benzofuran-2-ylmethylenethiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 246 (M+1); Rt.=3.65 min, mp=265-266° C.

Example 217 General Procedure (C)5-[3-(4-Dimethylaminophenyl)allylidene]thiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 276 (M+1); Rt.=3.63, mp=259-263° C.

¹H-NMR: (DMSO-d₆) δ=12.3 (1H, broad); 7.46 (2H, d); 7.39 (1H, d); 7.11(1H, d); 6.69 (2H, d); 6.59 (1H, dd); 2.98 (3H, s).

Example 218 General Procedure (C)5-(2-Methyl-3-phenylallylidene)thiazolidine-2,4-dione

Mp: 203-210° C.

HPLC-MS (Method C): m/z: 246 (M+1); Rt=3.79 min.

Example 219 General Procedure (C)5-(2-Chloro-3-phenylallylidene)thiazolidine-2,4-dione

Mp: 251-254° C.

HPLC-MS (Method C): m/z: 266 (M+1; Rt=3.90 min

Example 220 General Procedure (C)5-(2-Oxo-1,2-dihydroquinolin-3-ylmethylene)thiazolidine-2,4-dione

Mp: 338-347° C.

HPLC-MS (Method C): m/z: 273 (M+1); Rt.=2.59 min.

Example 221 General Procedure (C)5-(2,4,6-Tribromo-3-hydroxybenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 459 (M+1); Rt.=3.65 min.

Example 222 General Procedure (C)5-(5-Bromo-2-methylindol-3-ylmethylene)thiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 339 (M+1); Rt=3.37 min.

Example 223 General Procedure (C)5-(7-Bromo-2-methylindol-3-ylmethylene)thiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 319 (M+1); Rt=3.48 min.

Example 224 General Procedure (C)5-(6-Bromoindol-3-ylmethylene)thiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 325 (M+1); Rt=3.54 min.

Example 225 General Procedure (C)5-(8-Methyl-2-oxo-1,2-dihydroquinolin-3-ylmethylene)thiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 287 (M+1); Rt=2.86 min.

Example 226 General Procedure (C)5-(6-Methoxy-2-oxo-1,2-dihydroquinolin-3-ylmethylene)thiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 303 (M+1); Rt=2.65 min.

Example 227 General Procedure (C)5-Quinolin-3-ylmethylenethiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 257 (M+1); Rt=2.77 min.

Example 228 General Procedure (C)5-(8-Hydroxyquinolin-2-ylmethylene)thiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 273 (M+1); Rt=3.44 min.

Example 229 General Procedure (C)5-Quinolin-8-ylmethylenethiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 257 (M+1); Rt=3.15 min.

Example 230 General Procedure (C)5-(1-Bromo-6-methoxynaphthalen-2-ylmethylene)thiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 366 (M+1); Rt=4.44 min.

Example 231 General Procedure (C)5-(6-Methyl-2-oxo-1,2-dihydroquinolin-3-ylmethylene)thiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 287 (M+1); Rt.=2.89 min.

Example 232 General Procedure (D)5-(2,6-Dichloro-4-dibenzylaminobenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method C): m/z: 469 (M+1); Rt=5.35 min.

Other preferred compounds include

3′,5′-Dichloro-4′-(2,4-dioxothiazolidin-5-ylidenemethyl)biphenyl-4-carboxylicacid

The following compounds are commercially available and may be preparedusing general procedures (B) and/or (C).

Example 233 5-(5-Bromo-1H-indol-3-ylmethylene)thiazolidine-2,4-dione

Example 234 5-Pyridin-4-ylmethylenethiazolidine-2,4-dione

Example 235 5-(3-Bromo-4-methoxybenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method A):

Example 236 5-(3-Nitrobenzylidene)thiazolidine-2,4-dione

HPLC-MS (Method A):

Example 237 5-Cyclohexylidene-1,3-thiazolidine-2,4-dione

HPLC-MS (Method A):

Example 238 5-(3,4-Dihydroxybenzylidene)thiazolidine-2,4-dione

Example 239 5-(3-Ethoxy-4-hydroxybenzylidene)thiazolidine-2,4-dione

Example 2405-(4-Hydroxy-3-methoxy-5-nitrobenzylidene)thiazolidine-2,4-dione

Example 241 5-(3-Ethoxy-4-hydroxybenzylidene)thiazolidine-2,4-dione

Example 242 5-(4-Hydroxy-3,5-dimethoxybenzylidene)thiazolidine-2,4-dione

Example 2435-(3-Bromo-5-ethoxy-4-hydroxybenzylidene)thiazolidine-2,4-dione

Example 2445-(3-Ethoxy-4-hydroxy-5-nitrobenzylidene)thiazolidine-2,4-dione

Example 245

Example 246

Example 247

Example 248

Example 249

Example 250

Example 251

Example 252

Example 253

Example 254

Example 255

Example 256

Example 257 5-(3-Hydroxy-5-methyl-phenylamino)-thiazolidine-2,4-dione

Example 258

Example 259

Example 260

Example 261

Example 262

Example 263

Example 264

Example 265

Example 266

Example 267

Example 268

Example 269

Example 270

Example 271

Example 272

Example 273

Example 274

Example 275

Example 276

Example 277

Example 278

Example 279

Example 280

Example 281

General Procedure (D) for Preparation of Compounds of General FormulaI₃:

-   -   wherein X, Y, R¹⁰ are as defined above,    -   n is 1 or 3-20,    -   E is arylene or heterarylene (including up to four optional        substituents, R¹³, R¹⁴, R¹⁵, and R^(15A) as defined above),    -   R′ is a standard carboxylic acid protecting group, such as        C₁-C₆-alkyl or benzyl and Lea is a leaving group, such as        chloro, bromo, iodo, methanesulfonyloxy, toluenesulfonyloxy or        the like.

Step 1 is an alkylation of a phenol moiety. The reaction is preformed byreacting R¹⁰—C(═O)-E-OH with an ω-bromo-alkane-carboxylic acid ester (ora synthetic equivalent) in the presence of a base such as sodium orpotassium carbonate, sodium or potassium hydroxide, sodium hydride,sodium or potassium alkoxide in a solvent, such as DMF, NMP, DMSO,acetone, acetonitrile, ethyl acetate or isopropyl acetate. The reactionis performed at 20-160° C., usually at room temperature, but when thephenol moiety has one or more substituents heating to 50° C. or more canbe beneficial, especially when the substituents are in the orthoposition relatively to the phenol. This will readily be recognised bythose skilled in the art.

Step 2 is a hydrolysis of the product from step 1.

Step 3 is similar to general procedure (B) and (C).

This general procedure (D) is further illustrated in the followingexamples:

Example 282 General Procedure (D)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyric acid

Step 1:

A mixture of 4-hydroxybenzaldehyde (9.21 g, 75 mmol), potassiumcarbonate (56 g, 410 mmol) and 4-bromobutyric acid ethyl ester (12.9 mL,90 mmol) in N,N-dimethylformamide (250 mL) was stirred vigorously for 16hours at room temperature. The mixture was filtered and concentrated invacuo to afford 19.6 g (100%) of 4-(4-formylphenoxy)butyric acid ethylester as an oil. ¹H-NMR (DMSO-d₆): δ 1.21 (3H, t), 2.05 (2H, p), 2.49(2H, t), 4.12 (4H, m), 7.13 (2H, d), 7.87 (2H, d), 9.90 (1H, s). HPLC-MS(Method A): m/z=237 (M+1); R_(t)=3.46 min.

Step 2:

4-(4-Formylphenoxy)butyric acid ethyl ester (19.6 g, 75 mmol) wasdissolved in methanol (250 mL) and 1N sodium hydroxide (100 mL) wasadded and the resulting mixture was stirred at room temperature for 16hours. The organic solvent was evaporated in vacuo (40° C., 120 mBar)and the residue was acidified with 1N hydrochloric acid (110 mL). Themixture was filtered and washed with water and dried in vacuo to afford14.3 g (91%) 4-(4-formylphenoxy)butyric acid as a solid. ¹H-NMR(DMSO-d₆): δ 1.99 (2H, p), 2.42 (2H, t), 4.13 (2H, t), 7.14 (2H, d),7.88 (2H, d), 9.90 (1H, s), 12.2 (1H, bs). HPLC-MS (Method A): m/z=209(M+1); R_(t)=2.19 min.

Step 3:

Thiazolidine-2,4-dione (3.55 g, 27.6 mmol), 4-(4-formylphenoxy)butyricacid (5.74 g, 27.6 mmol), anhydrous sodium acetate (11.3 g, 138 mmol)and acetic acid (100 mL) was refluxed for 16 h. After cooling, themixture was filtered and washed with acetic acid and water. Drying invacuo afforded 2.74 g (32%) of4-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyric acid as asolid.

¹H-NMR (DMSO-d₆): δ 1.97 (2H, p), 2.40 (2H, t), 4.07 (2H, t), 7.08 (2H,d), 7.56 (2H, d), 7.77 (1H, s), 12.2 (1H, bs), 12.5 (1H, bs); HPLC-MS(Method A): m/z: 308 (M+1); Rt=2.89 min.

Example 283 General Procedure (D)[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetic acid

Step 3:

Thiazolidine-2,4-dione (3.9 g, 33 mmol), 3-formylphenoxyacetic acid (6.0g, 33 mmol), anhydrous sodium acetate (13.6 g, 165 mmol) and acetic acid(100 mL) was refluxed for 16 h. After cooling, the mixture was filteredand washed with acetic acid and water. Drying in vacuo afforded 5.13 g(56%) of [3-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]acetic acid asa solid.

¹H-NMR (DMSO-d₆): δ 4.69 (2H, s), 6.95 (1H, dd), 7.09 (1H, t), 7.15 (1H,d), 7.39 (1H, t), 7.53 (1H, s); HPLC-MS (Method A): m/z=280 (M+1) (poorionisation); R_(t)=2.49 min.

The compounds in the following examples were similarly prepared.

Example 284 General Procedure (D)3-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenyl]acrylic acid

¹H-NMR (DMSO-d₆): δ 6.63 (1H, d), 7.59-7.64 (3H, m), 7.77 (1H, s), 7.83(2H, m).

Example 285 General Procedure (D)[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetic acid

Triethylamine salt: ¹H-NMR (DMSO-d₆): δ 4.27 (2H, s), 6.90 (2H, d), 7.26(1H, s), 7.40 (2H, d).

Example 286 General Procedure (D)4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzoic acid

Example 287 General Procedure (D)3-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzoic acid

¹H-NMR (DMSO-d₆): δ 7.57 (1H, s), 7.60 (1H, t), 7.79 (1H, dt), 7.92 (1H,dt), 8.14 (1H, t).

Example 288 General Procedure (D)4-[2-Chloro-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyric acid

¹H-NMR (DMSO-d₆): δ 2.00 (2H, p), 2.45 (2H, t), 4.17 (2H, t), 7.31 (1H,d), 7.54 (1H, dd), 7.69 (1H, d), 7.74 (1H, s), 12.2 (1H, bs), 12.6 (1H,bs). HPLC-MS (Method A): m/z: 364 (M+23); Rt=3.19 min.

Example 289 General Procedure (D)4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyric acid

¹H-NMR (DMSO-d₆): δ 1.99 (2H, p), 2.46 (2H, t), 4.17 (2H, t), 7.28 (1H,d), 7.57 (1H, dd), 7.25 (1H, s), 7.85 (1H, d), 12.2 (1H, bs), 12.6 (1H,bs). HPLC-MS (Method A): m/z: 410 (M+23); Rt=3.35 min.

Example 290 General Procedure (D)4-[2-Bromo-4-(4-oxo-2-thioxothiazolidin-5-ylidenemethyl)phenoxy]butyricacid

¹H-NMR (DMSO-d₆): δ 1.99 (2H, p), 2.45 (2H, t), 4.18 (2H, t), 7.28 (1H,d), 7.55 (1H, dd), 7.60 (1H, s), 7.86 (1H, d), 12.2 (1H, bs), 13.8 (1H,bs). HPLC-MS (Method A): m/z: 424 (M+23); Rt=3.84 min.

HPLC-MS (Method A): m/z: 424 (M+23); Rt=3.84 min

Example 291 General Procedure (D)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyricacid

¹H-NMR (DMSO-d₆): δ 2.12 (2H, p), 2.5 (below DMSO), 4.28 (2H, t), 7.12(1H, d), 7.6-7.7 (3H, m), 8.12 (1H, d), 8.31 (1H, d), 8.39 (1H, s), 12.2(1H, bs, 12.6 (1H, bs). HPLC-MS (method A): m/z: 380 (M+23); Rt=3.76min.

Example 292 General Procedure (D)5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoicacid

HPLC-MS (Method A): m/z: 394 (M+23); Rt=3.62 min. ¹H-NMR (DMSO-d₆): δ1.78 (2H, m), 1.90 (2H, m), 2.38 (2H, t), 4.27 (2H, t), 7.16 (1H, d),7.6-7.75 (3H, m), 8.13 (1H, d), 8.28 (1H, d), 8.39 (1H, s), 12.1 (1H,bs), 12.6 (1H, bs).

Example 2935-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoicacid

5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)-naphthalen-1-yloxy]pentanoicacid (example 292, 185 mg, 0.5 mmol) was treated with an equimolaramount of bromine in acetic acid (10 mL). Stirring at RT for 14 daysfollowed by evaporation to dryness afforded a mixture of the brominatedcompound and unchanged starting material. Purification by preparativeHPLC on a C18 column using acetonitrile and water as eluent afforded 8mg of the title compound.

HPLC-MS (Method C): m/z: 473 (M+23), Rt.=3.77 min

Example 2944-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyricacid

Starting with4-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)-naphthalen-1-yloxy]-butyricacid (example 291, 0.5 mmol) using the same method as in example 293afforded 66 mg of the title compound.

HPLC-MS (Method C): m/z: 459 (M+23); Rt.=3.59 min.

Example 295 General Procedure (D)[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]acetic acid

¹H-NMR (DMSO-d₆): δ 4.90 (2H, s), 7.12 (1H, d), 7.52 (1H, dd), 7.65 (1H,s) 7.84 (1H, d). HPLC-MS (Method A): m/z: not observed; Rt=2.89 min.

Example 296 General Procedure (D)4-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyric acid

¹H-NMR (DMSO-d₆): δ 1.98 (2H, p), 2.42 (2H, t), 4.04 (2H, t), 7.05 (1H,dd), 7.15 (2H, m), 7.45 (1H, t), 7.77 (1H, s), 12.1 (1H, bs), 12.6 (1H,bs). HPLC-MS (Method A): m/z: 330 (M+23); Rt=3.05 min.

Example 297 General Procedure (D)[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)-3-methoxyphenoxy]acetic acid

HPLC-MS (Method B): m/z: 310 (M+1); Rt=3.43 min.

Example 298 General Procedure (D)[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]acetic acid

HPLC-MS (Method A): m/z: 330 (M+1); Rt=3.25 min.

Example 299 General Procedure (D)8-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalene-1-carboxylic acid

HPLC-MS (Method A): m/z: 299 (M+1); Rt=2.49 min.

Example 300 General Procedure (D)[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indol-1-yl]acetic acid

HPLC-MS (Method A): m/z: 303 (M+1); Rt=2.90 min.

Preparation of Starting Material:

3-Formylindol (10 g, 69 mmol) was dissolved in N,N-dimethylformamide(100 mL) and under an atmosphere of nitrogen and with external cooling,keeping the temperature below 15° C., sodium hydride (60% in mineraloil, 3.0 g, 76 mmol) was added in portions. Then a solution of ethylbromoacetate (8.4 mL, 76 mmol) in N,N-dimethylformamide (15 mL) wasadded dropwise over 30 minutes and the resulting mixture was stirred atroom temperature for 16 hours. The mixture was concentrated in vacuo andthe residue was partitioned between water (300 mL) and ethyl acetate(2×150 mL). The combined organic extracts were washed with a saturatedaqueous solution of ammonium chloride (100 mL), dried (MgSO₄) andconcentrated in vacuo to afford 15.9 g (quant.) of(3-formylindol-1-yl)acetic acid ethyl ester as an oil.

¹H-NMR (CDCl₃): δ_(H)=1.30 (3H, t), 4.23 (2H, q), 4.90 (2H, s), 7.3 (3H,m), 7.77 (1H, s), 8.32 (1H, d), 10.0 (1H, s).

(3-Formylindol-1-yl)acetic acid ethyl ester (15.9 g 69 mmol) wasdissolved in 1,4-dioxane (100 mL) and 1N sodium hydroxide (10 mL) wasadded and the resulting mixture was stirred at room temperature for 4days. Water (500 mL) was added and the mixture was washed with diethylether (150 mL). The aqueous phase was acidified with 5N hydrochloricacid and extracted with ethyl acetate (250+150 mL). The combined organicextracts were dried (MgSO₄) and concentrated in vacuo to afford 10.3 g(73%) of (3-formylindol-1-yl)acetic acid as a solid.

¹H-NMR (DMSO-d₆): δ_(H)=5.20 (2H, s), 7.3 (2H, m), 7.55 (1H, d), 8.12(1H, d), 8.30 (1H, s), 9.95 (1H, s), 13.3 (1H, bs).

Example 301 General Procedure (D)3-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indol-1-yl]propionic acid

HPLC-MS (Method A): m/z: 317 (M+1); Rt=3.08 min.

Preparation of Starting Material:

A mixture of 3-formylindol (10 g, 69 mmol), ethyl 3-bromopropionate(10.5 mL, 83 mmol) and potassium carbonate (28.5 g, 207 mmol) andacetonitrile (100 mL) was stirred vigorously at refux temperature for 2days. After cooling, the mixture was filtered and the filtrate wasconcentrated in vacuo to afford 17.5 g (quant.) of3-(3-formylindol-1-yl)propionic acid ethyl ester as a solid.

¹H-NMR (DMSO-d₆): δ_(H)=1.10 (3H, t), 2.94 (2H, t), 4.02 (2H, q), 4.55(2H, t), 7.3 (2H, m), 7.67 (1H, d), 8.12 (1H, d), 8.30 (1H, s), 9.90(1H, s).

3-(3-Formylindol-1-yl)propionic acid ethyl ester (17.5 g 69 mmol) washydrolysed as described above to afford 12.5 g (83%) of3-(3-formylindol-1-yl)propionic acid as a solid.

¹H-NMR (DMSO-d₆): δ_(H)=2.87 (2H, t), 4.50 (2H, t), 7.3 (2H, m), 7.68(1H, d), 8.12 (1H, d), 8.31 (1H, s), 9.95 (1H, s), 12.5 (1H, bs).

Example 302 General Procedure (D){5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzylidene]-4-oxo-2-thioxothiazolidin-3-yl}aceticacid

HPLC-MS (Method A): m/z: 429 (M+23); Rt=3.89 min.

Example 303 General Procedure (D)6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxyoctanoic acid

HPLC-MS (Method C): m/z: 436 (M+23); Rt.=4.36 min

The intermediate aldehyde for this compound was prepared by a slightlymodified procedure: 6-Hydroxynaphthalene-2-carbaldehyde (1.0 g, 5.8mmol) was dissolved in DMF (10 mL) and sodium hydride 60% (278 mg) wasadded and the mixture stirred at RT for 15 min. 8-Bromooctanoic acid(0.37 g, 1.7 mmol) was converted to the sodium salt by addition ofsodium hydride 60% and added to an aliquot (2.5 mL) of the abovenaphtholate solution and the resulting mixture was stirred at RT for 16hours. Aqueous acetic acid (10%) was added and the mixture was extracted3 times with diethyl ether. The combined organic phases were dried withMgSO₄ and evaporated to dryness affording 300 mg of8-(6-formylnaphthalen-2-yloxy)octanoic acid.

HPLC-MS (Method C): m/z 315 (M+1); Rt.=4.24 min.

Example 304 General Procedure (D)12-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]dodecanoicacid

HPLC-MS (Method C): m/z: 492 (M+23); Rt.=5.3 min.

The intermediate aldehyde was prepared similarly as described in example303.

Example 305 General Procedure (D)11-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]undecanoicacid

HPLC-MS (Method C): m/z: 478 (M+23); Rt.=5.17 min.

The intermediate aldehyde was prepared similarly as described in example303.

Example 306 General Procedure (D)15-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]pentadecanoicacid

HPLC-MS (Method C): m/z: 534 (M+23); Rt.=6.07 min.

The intermediate aldehyde was prepared similarly as described in example303.

Example 307 General Procedure (D)6-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]hexanoicacid

HPLC-MS (Method C): m/z: 408 (M+23); Rt.=3.71 min.

Example 308 General Procedure (D)4-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]butyricacid

HPLC-MS (Method C): m/z: 380 (M+23); Rt.=3.23 min.

Example 309 General Procedure (D)6-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]hexanoicacid ethyl ester

HPLC-MS (Method C): m/z: 436 (M+23); Rt.=4.64 min.

Example 310 General Procedure (D)4-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]butyricacid ethyl ester

HPLC-MS (Method C): m/z: 408 (M+23); Rt.=4.28 min.

Example 311N-(3-Aminopropyl)-4-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)-naphthalen-1-yloxy]-butyramide

To a mixture of4-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyricacid (example 291, 5.9 g, 16.5 mmol) and 1-hydroxybenzotriazole (3.35 g,24.8 mmol) in DMF (60 mL) was added1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (4.75 g,24.8 mmol) and the resulting mixture was stirred at room temperature for2 hours. N-(3-aminopropylcarbamic acid tert-butyl ester (3.45 g, 19.8mmol) was added and the resulting mixture was stirred at roomtemperature for 16 hours. The mixture was concentrated in vacuo andethyl acetate and dichloromethane were added to the residue. The mixturewas filtered, washed with water and dried in vacuo to afford 4.98 g(59%) of(3-{4-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyrylamino}propyl)carbamicacid tert-butyl ester.

HPLC-MS (Method C): m/z: 515 (M+1); Rt=3.79 min.

(3-{4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyrylamino}-propyl)carbamicacid tert-butyl ester (4.9 g, 9.5 mmol) was added dichloromethane (50mL) and trifluoroacetic acid (50 mL) and the resulting mixture wasstirred at room temperature for 45 minutes. The mixture was concentratedin vacuo and co-evaporated with toluene. To the residue was added ethylacetate (100 mL) and the mixture was filtered and dried in vacuo toafford the title compound as the trifluoroacetic acid salt.

HPLC-MS (Method C): m/z: 414 (M+1); Rt=2.27 min.

Preferred Compounds of the Invention Includes:

The following compounds are commercially available and may be preparedaccording to general procedure (D):

Example 312

Example 313

Example 314

Example 315

Example 316

Example 317

Example 318

The following salicylic acid derivatives do all bind to the His B10 Zn²⁺site of the insulin hexamer:

Example 319 Salicylic Acid

Example 320 Thiosalicylic Acid (or: 2-Mercaptobenzoic acid)

Example 321 2-Hydroxy-5-nitrobenzoic acid

Example 322 3-Nitrosalicyclic acid

Example 323 5,5′-Methylenedisalicylic acid

Example 324 2-Amino-5-trifluoromethylbenzoesyre

Example 325 2-Amino-4-chlorobenzoic acid

Example 326 2-Amino-5-methoxybenzoesyre

Example 327

Example 328

Example 329

Example 330

Example 331

Example 332

Example 333 5-Iodosalicylic acid

Example 334 5-Chlorosalicylic acid

Example 335 1-Hydroxy-2-naphthoic acid

Example 336 3,5-Dihydroxy-2-naphthoic acid

Example 337 3-Hydroxy-2-naphthoic acid

Example 338 3,7-Dihydroxy-2-naphthoic acid

Example 339 2-Hydroxybenzo[a]carbazole-3-carboxylic acid

Example 340 7-Bromo-3-hydroxy-2-naphthoic acid

This compound was prepared according to Murphy et al., J. Med. Chem.1990, 33, 171-8.

HPLC-MS (Method A): m/z: 267 (M+1), Rt:=3.78 min.

Example 341 1,6-Dibromo-2-hydroxynaphthalene-3-carboxylic acid

This compound was prepared according to Murphy et al., J. Med. Chem.1990, 33, 171-8. HPLC-MS (Method A): m/z: 346 (M+1); Rt:=4.19 min.

Example 342 7-Formyl-3-hydroxynaphthalene-2-carboxylic acid

A solution of 7-bromo-3-hydroxynaphthalene-2-carboxylic acid (15.0 g,56.2 mmol) (example 340) in tetrahydrofuran (100 mL) was added to asolution of lithium hydride (893 mg, 112 mmol) in tetrahydrofuran (350mL). After 30 minutes stirring at room temperature, the resultingsolution was heated to 50° C. for 2 minutes and then allowed to cool toambient temperature over a period of 30 minutes. The mixture was cooledto −78° C., and butyllithium (1.6 M in hexanes, 53 mL, 85 mmol) wasadded over a period of 15 minutes. N,N-Dimethylformamide (8.7 mL, 8.2 g,112 mmol) was added after 90 minutes additional stirring. The coolingwas discontinued, and the reaction mixture was stirred at roomtemperature for 17 hours before it was poured into 1 N hydrochloric acid(aq.) (750 mL). The organic solvents were evaporated in vacuo, and theresulting precipitate was filtered off and rinsed with water (3×100 mL)to yield the crude product (16.2 g). Purification on silica gel(dichloromethane/methanol/acetic acid=90:9:1) furnished the titlecompound as a solid.

¹H-NMR (DMSO-d₆): δ 11.95 (1H, bs), 10.02 (1H, s), 8.61 (1H, s), 8.54(1H, s), 7.80 (2H, bs), 7.24 (1H, s); HPLC-MS (Method (A)): m/z: 217(M+1); Rt=2.49 min.

Example 343 3-Hydroxy-7-methoxy-2-naphthoic acid

Example 344 4-Amino-2-hydroxybenzoic acid

Example 345 5-Acetylamino-2-hydroxybenzoic acid

Example 346 2-Hydroxy-5-methoxybenzoic acid

The following compounds were prepared as described below:

Example 347 4-Bromo-3-hydroxynaphthalene-2-carboxylic acid

3-Hydroxynaphthalene-2-carboxylic acid (3.0 g, 15.9 mmol) was suspendedin acetic acid (40 mL) and with vigorous stirring a solution of bromine(817 μL, 15.9 mmol) in acetic acid (10 mL) was added drop wise during 30minutes. The suspension was stirred at room temperature for 1 hour,filtered and washed with water. Drying in vacuo afforded 3.74 g (88%) of4-bromo-3-hydroxynaphthalene-2-carboxylic acid as a solid.

¹H-NMR (DMSO-d₆): δ 7.49 (1H, t), 7.75 (1H, t), 8.07 (2H, “t”), 8.64(1H, s). The substitution pattern was confirmed by a COSY experiment,showing connectivities between the 3 (4 hydrogen) “triplets”. HPLC-MS(Method A): m/z: 267 (M+1); Rt=3.73 min.

Example 348 3-Hydroxy-4-iodonaphthalene-2-carboxylic acid

3-Hydroxynaphthalene-2-carboxylic acid (0.5 g, 2.7 mmol) was suspendedin acetic acid (5 mL) and with stirring iodine monochloride (135 μL, 2.7mml) was added. The suspension was stirred at room temperature for 1hour, filtered and washed with water. Drying afforded 0.72 g (85%) of4-iodo-3-hydroxynaphthalene-2-carboxylic acid as a solid.

¹H-NMR (DMSO-d₆): δ 7.47 (1H, t), 7.73 (1H, t), 7.98 (1H, d), 8.05 (1H,d), 8.66 (1H, s). HPLC-MS (Method A): m/z: 315 (M+1); Rt=3.94 min.

Example 349 2-Hydroxy-5-[(4-methoxyphenylamino)methyl]benzoic acid

p-Anisidine (1.3 g, 10.6 mmol) was dissolved in methanol (20 mL) and5-formylsalicylic acid (1.75 g, 10.6 mmol) was added and the resultingmixture was stirred at room temperature for 16 hours. The solid formedwas isolated by filtration, re-dissolved in N-methyl pyrrolidone (20 mL)and methanol (2 mL). To the mixture was added sodium cyanoborohydride(1.2 g) and the mixture was heated to 70° C. for 3 hours. To the cooledmixture was added ethyl acetate (100 mL) and the mixture was extractedwith water (100 mL) and saturated aqueous ammonium chloride (100 mL).The combined aqueous phases were concentrated in vacuo and a 2 g aliquotwas purified by SepPac chromatography eluting with mixtures ofaetonitrile and water containing 0.1% trifluoroacetic acid to afford thetitle compound.

HPLC-MS (Method A): m/z: 274 (M+1); Rt=1.77 min.

¹H-NMR (methanol-d₄): δ 3.82 (3H, s), 4.45 (2H, s), 6.96 (1H, d), 7.03(2H, d), 7.23 (2H, d), 7.45 (1H, dd), 7.92 (1H, d).

Example 350 2-Hydroxy-5-(4-methoxyphenylsulfamoyl)benzoic acid

A solution of 5-chlrosulfonylsalicylic acid (0.96 g, 4.1 mmol) indichloromethane (20 mL) and triethylamine (1.69 mL, 12.2 mmol) was addedp-anisidine (0.49 g, 4.1 mmol) and the resulting mixture was stirred atroom temperature for 16 hours. The mixture was added dichloromethane (50mL) and was washed with water (2×100 mL). Drying (MgSO₄) of the organicphase and concentration in vacuo afforded 0.57 g crude product.Purification by column chromatography on silica gel eluting first withethyl acetate:heptane (1:1) then with methanol afforded 0.1 g of thetitle compound.

HPLC-MS (Method A): m/z: 346 (M+23); Rt=2.89 min.

¹H-NMR (DMSO-d₆): δ 3.67 (3H, s), 6.62 (1H, d), 6.77 (2H, d), 6.96 (2H,d), 7.40 (1H, dd), 8.05 (1H, d), 9.6 (1H, bs).

General Procedure (E) for Preparation of Compounds of General FormulaI₄:

wherein Lea is a leaving group such as Cl, Br, I or OSO₂CF₃, R ishydrogen or C₁-C₆-alkyl, optionally the two R-groups may together form a5-8 membered ring, a cyclic boronic acid ester, and T is as definedabove.

An analogous chemical transformation has previously been described inthe literature (Bumagin et al., Tetrahedron, 1997, 53, 14437-14450). Thereaction is generally known as the Suzuki coupling reaction and isgenerally performed by reacting an aryl halide or triflate with anarylboronic acid or a heteroarylboronic acid in the presence of apalladium catalyst and a base such as sodium acetate, sodium carbonateor sodium hydroxide. The solvent can be water, acetone, DMF, NMP, HMPA,methanol, ethanol toluene or a mixture of two or more of these solvents.The reaction is performed at room temperature or at elevatedtemperature.

The general procedure (E) is further illustrated in the followingexample:

Example 351 General Procedure (E)7-(4-Acetylphenyl)-3-hydroxynaphthalene-2-carboxylic acid

To 7-bromo-3-hydroxynaphthalene-2-carboxylic acid (100 mg, 0.37 mmol)(example 340) was added a solution of 4-acetylphenylboronic acid (92 mg,0.56 mmol) in acetone (2.2 mL) followed by a solution of sodiumcarbonate (198 mg, 1.87 mmol) in water (3.3 mL). A suspension ofpalladium(II) acetate (4 mg, 0.02 mmol) in acetone (0.5 mL) was filteredand added to the above solution. The mixture was purged with N₂ andstirred vigorously for 24 hours at room temperature. The reactionmixture was poured into 1 N hydrochloric acid (aq.) (60 mL) and theprecipitate was filtered off and rinsed with water (3×40 mL). The crudeproduct was dissolved in acetone (25 mL) and dried with magnesiumsulfate (1 h). Filtration followed by concentration furnished the titlecompound as a solid (92 mg). ¹H-NMR (DMSO-d₆): δ 12.60 (1H, bs), 8.64(1H, s), 8.42 (1H, s), 8.08 (2H, d), 7.97 (2H, d), 7.92 (2H, m), 7.33(1H, s), 2.63 (3H, s); HPLC-MS (Method (A): m/z: 307 (M+1); Rt=3.84 min.

The compounds in the following examples were prepared in a similarfashion. Optionally, the compounds can be further purified byrecrystallization from e.g. ethanol or by chromatography.

Example 352 General Procedure (E)3-Hydroxy-7-(3-methoxyphenyl)naphthalene-2-carboxylic acid

HPLC-MS (Method (A)): m/z: 295 (M+1); Rt=4.60 min.

Example 353 General Procedure (E)3-Hydroxy-7-phenylnaphthalene-2-carboxylic acid

HPLC-MS (Method (A)): m/z: 265 (M+1); Rt=4.6 min.

Example 354 General Procedure (E)3-Hydroxy-7-p-tolylnaphthalene-2-carboxylic acid

HPLC-MS (Method (A)): m/z: 279 (M+1); Rt=4.95 min.

Example 355 General Procedure (E)7-(4-Formylphenyl)-3-hydroxynaphthalene-2-carboxylic acid

HPLC-MS (Method (A)): m/z: 293 (M+1); Rt=4.4 min.

Example 356 General Procedure (E)6-Hydroxy-[1,2]binaphthalenyl-7-carboxylic acid

HPLC-MS (Method (A)): m/z: 315 (M+1); Rt=5.17 min.

Example 357 General Procedure (E)7-(4-Carboxy-phenyl)-3-hydroxynaphthalene-2-carboxylic acid

HPLC-MS (Method (A)): m/z: 309 (M+1); Rt=3.60 min.

Example 358 General Procedure (E)7-Benzofuran-2-yl-3-hydroxynaphthalene-2-carboxylic acid

HPLC-MS (Method (A)): m/z: 305 (M+1); Rt=4.97 min.

Example 359 General Procedure (E)3-Hydroxy-7-(4-methoxyphenyl)-naphthalene-2-carboxylic acid

HPLC-MS (Method (A)): m/z: 295 (M+1); Rt=4.68 min.

Example 360 General Procedure (E)7-(3-Ethoxyphenyl)-3-hydroxynaphthalene-2-carboxylic acid

HPLC-MS (Method (A)): m/z: 309 (M+1); Rt=4.89 min.

Example 361 General Procedure (E)7-Benzo[1,3]dioxol-5-yl-3-hydroxynaphthalene-2-carboxylic acid

HPLC-MS (Method (A)): m/z: 309 (M+1); Rt=5.61 min.

Example 362 General Procedure (E)7-Biphenyl-3-yl-3-hydroxynaphthalene-2-carboxylic acid

HPLC-MS (Method (A)): m/z: 341 (M+1); Rt=5.45 min.

General Procedure (F) for Preparation of Compounds of General FormulaI₅:

wherein R³⁰ is hydrogen or C₁-C₆-alkyl and T is as defined above

This general procedure (F) is further illustrated in the followingexample:

Example 363 General Procedure (F)3-Hydroxy-7-[(4-(2-propyl)phenylamino)methyl]naphthalene-2-carboxylicacid

7-Formyl-3-hydroxynaphthalene-2-carboxylic acid (40 mg, 0.19 mmol)(example 342) was suspended in methanol (300 μL). Acetic acid (16 μL, 17mg, 0.28 mmol) and 4-(2-propyl)aniline (40 μL, 40 mg, 0.30 mmol) wereadded consecutively, and the resulting mixture was stirred vigorously atroom temperature for 2 hours. Sodium cyanoborohydride (1.0 M intetrahydrofuran, 300 μL, 0.3 mmol) was added, and the stirring wascontinued for another 17 hours. The reaction mixture was poured into 6 Nhydrochloric acid (aq.) (6 mL), and the precipitate was filtered off andrinsed with water (3×2 mL) to yield the title compound (40 mg) as itshydrochloride salt. No further purification was necessary.

¹H-NMR (DMSO-d₆): δ 10.95 (1H, bs), 8.45 (1H, s), 7.96 (1H, s), 7.78(1H, d), 7.62 (1H, d), 7.32 (1H, s), 7.13 (2H, bd), 6.98 (2H, bd), 4.48(2H, s), 2.79 (1H, sept), 1.14 (6H, d); HPLC-MS (Method (A)): m/z: 336(M+1); Rt=3.92 min.

The compounds in the following examples were made using this generalprocedure (F).

Example 364 General Procedure (F)7-{[(4-Bromophenyl)amino]methyl}-3-hydroxynaphthalene-2-carboxylic acid

HPLC-MS (Method C): m/z: 372 (M+1); Rt=4.31 min.

Example 365 General Procedure (F)7-{[(3,5-Dichlorophenyl)amino]methyl}-3-hydroxynaphthalene-2-carboxylicacid

HPLC-MS (Method C): m/z: 362 (M+1); Rt=4.75 min.

Example 366 General Procedure (F)7-{[(Benzothiazol-6-yl)amino]methyl}-3-hydroxynaphthalene-2-carboxylicacid

HPLC-MS (Method C): m/z 351 (M+1); Rt=3.43 min.

Example 367 General Procedure (F)3-Hydroxy-7-{[(quinolin-6-yl)amino]methyl}naphthalene-2-carboxylic acid

HPLC-MS (Method C): m/z: 345 (M+1); Rt=2.26 min.

Example 368 General Procedure (F)3-Hydroxy-7-{[(4-methoxyphenyl)amino]methyl}naphthalene-2-carboxylicacid

HPLC-MS (Method C): m/z: 324 (M+1); Rt=2.57 min.

Example 369 General Procedure (F)7-{[(2,3-Dihydrobenzofuran-5-ylmethyl)amino]methyl}-3-hydroxynaphthalene-2-carboxylicacid

HPLC-MS (Method C): m/z: 350 (M+1); Rt=2.22 min.

Example 370 General Procedure (F)7-{[(4-Chlorobenzyl)amino]methyl}-3-hydroxynaphthalene-2-carboxylic acid

HPLC-MS (Method C): m/z: 342 (M+1); Rt=2.45 min.

Example 371 General Procedure (F)3-Hydroxy-7-{[(naphthalen-1-ylmethyl)amino]methyl}naphthalene-2-carboxylicacid

HPLC-MS (Method C): m/z: 357 (M+1); Rt=2.63 min.

Example 372 General Procedure (F)7-{[(Biphenyl-2-ylmethyl)amino]methyl}-3-hydroxynaphthalene-2-carboxylicacid

HPLC-MS (Method C): m/z: 384 (M+1); Rt=2.90 min.

Example 373 General Procedure (F)3-Hydroxy-7-{[(4-phenoxybenzyl)amino]methyl}naphthalene-2-carboxylicacid

HPLC-MS (Method C): m/z: 400 (M+1); Rt=3.15 min.

Example 374 General Procedure (F)3-Hydroxy-7-{[(4-methoxybenzyl)amino]methyl}naphthalene-2-carboxylicacid

HPLC-MS (Method C): m/z: 338 (M+1); Rt=2.32 min.

General Procedure (G) for Preparation of Compounds of General FormulaI₆:

wherein T is as defined above and the moiety (C₁-C₆-alkanoyl)₂O is ananhydride.

The general procedure (G) is illustrated by the following example:

Example 375 General Procedure (G)N-Acetyl-3-hydroxy-7-[(4-(2-propyl)phenylamino)methyl]naphthalene-2-carboxylicacid

3-Hydroxy-7-[(4-(2-propyl)phenylamino)methyl]naphthalene-2-carboxylicacid (25 mg, 0.07 mmol) (example 363) was suspended in tetrahydrofuran(200 μL). A solution of sodium hydrogencarbonate (23 mg, 0.27 mmol) inwater (200 μL) was added followed by acetic anhydride (14 μL, 15 mg,0.15 mmol). The reaction mixture was stirred vigorously for 65 hours atroom temperature before 6 N hydrochloric acid (4 mL) was added. Theprecipitate was filtered off and rinsed with water (3×1 mL) to yield thetitle compound (21 mg). No further purification was necessary.

¹H-NMR (DMSO-d₆): δ 10.96 (1H, bs), 8.48 (1H, s), 7.73 (1H, s), 7.72(1H, d), 7.41 (1H, dd), 7.28 (1H, s), 7.23 (2H, d), 7.18 (2H, d), 4.96(2H, s), 2.85 (1H, sept), 1.86 (3H, s), 1.15 (6H, d); HPLC-MS (Method(A)): m/z: 378 (M+1); Rt=3.90 min.

The compounds in the following examples were prepared in a similarfashion.

Example 376 General Procedure (G)N-Acetyl-7-{[(4-bromophenyl)amino]methyl}-3-hydroxynaphthalene-2-carboxylicacid

HPLC-MS (Method C): m/z: 414 (M+1); Rt=3.76 min.

Example 377 General Procedure (G)N-Acetyl-7-{[(2,3-dihydrobenzofuran-5-ylmethyl)amino]methyl}-3-hydroxynaphthalene-2-carboxylicacid

HPLC-MS (Method C): m/z: 392 (M+1); Rt=3.26 min.

Example 378 General Procedure (G)N-Acetyl-7-{[(4-chlorobenzyl)amino]methyl}-3-hydroxynaphthalene-2-carboxylicacid

HPLC-MS (Method C): m/z: 384 (M+1); Rt=3.67 min.

Example 379 5-(3-(Naphthalen-2-yloxymethyl)-phenyl)-1H-tetrazole

To a mixture of 2-naphthol (10 g, 0.07 mol) and potassium carbonate (10g, 0.073 mol) in acetone (150 mL), alpha-bromo-m-tolunitril (13.6 g,0.07 mol) was added in portions. The reaction mixture was stirred atreflux temperature for 2.5 hours. The cooled reaction mixture wasfiltered and evaporated in vacuo affording an oily residue (19 g) whichwas dissolved in diethyl ether (150 mL) and stirred with a mixture ofactive carbon and MgSO₄ for 16 hours. The mixture was filtered andevaporated in vacuo affording crude 18.0 g (100%) of3-(naphthalen-2-yloxymethyl)benzonitrile as a solid.

12 g of the above benzonitrile was recrystallised from ethanol (150 mL)affording 8.3 g (69%) of 3-(naphthalen-2-yloxymethyl)-benzonitrile as asolid.

M.p. 60-61° C.

Calculated for C₁₈H₁₃NO: C, 83.37%; H, 5.05%; N, 5.40%. Found C, 83.51%;H, 5.03%; N, 5.38%.

To a mixture of sodium azide (1.46 g, 22.5 mmol) and ammonium chloride(1.28 g, 24.0 mmol) in dry dimethylformamide (20 mL) under an atmosphereof nitrogen, 3-(naphthalen-2-yloxymethyl)-benzonitrile (3.9 g, 15 mmol)was added and the reaction mixture was stirred at 125° C. for 4 hours.The cooled reaction mixture was poured on to ice water (300 mL) andacidified to pH=1 with 1 N hydrochloric acid. The precipitate wasfiltered off and washed with water, dried at 100° C. for 4 hoursaffording 4.2 g (93%) of the title compound.

M.p. 200-202° C.

Calculated for C₁₈H₁₄N₄O: C, 71.51%; H, 4.67%; N, 18.54%. Found C,72.11%; H, 4.65%; N, 17.43%.

¹H NMR (400 MHz, DMSO-d₆) δ_(H) 5.36 (s, 2H), 7.29 (dd, 1H), 7.36 (dt,1H), 7.47 (m, 2H), 7.66 (t, 1H), 7.74 (d, 1H), 7.84 (m, 3H), 8.02 (d,1H), 8.22 (s, 1H).

Example 380 N-(3-(Tetrazol-5-yl)phenyl)-2-naphtoic acid amide

2-Naphtoic acid (10 g, 58 mmol) was dissolved in dichloromethane (100mL) and N,N-dimethylformamide (0.2 mL) was added followed by thionylchloride (5.1 ml, 70 mmol). The mixture was heated at reflux temperaturefor 2 hours. After cooling to room temperature, the mixture was addeddropwise to a mixture of 3-aminobenzonitril (6.90 g, 58 mmol) andtriethyl amine (10 mL) in dichloromethane (75 mL). The resulting mixturewas stirred at room temperature for 30 minutes. Water (50 mL) was addedand the volatiles was exaporated in vacuo. The resulting mixture wasfiltered and the filter cake was washed with water followed by heptane(2×25 mL). Drying in vacuo at 50° C. for 16 hours afforded 15.0 g (95%)of N-(3-cyanophenyl)-2-naphtoic acid amide.

M.p. 138-140° C.

The above naphthoic acid amide (10 g, 37 mmol) was dissolved inN,N-dimethylformamide (200 mL) and sodium azide (2.63 g, 40 mmol) andammonium chloride (2.16 g, 40 mmol) were added and the mixture heated at125° C. for 6 hours. Sodium azide (1.2 g) and ammonium chloride (0.98 g)were added and the mixture heated at 125° C. for 16 hours. Aftercooling, the mixture was poured into water (1.5 l) and stirred at roomtemperature for 30 minutes. The solid formed was filtered off, washedwith water and dried in vacuo at 50° C. for 3 days affording 9.69 g(84%) of the title compound as a solid which could be further purifiedby treatment with ethanol at reflux temperature.

¹H NMR (200 MHz, DMSO-d₆): δ_(H) 7.58-7.70 (3H), 7.77 (d, 1H), 8.04-8.13(m, 5H), 8.65 (d, 1H), 10.7 (s, 1H).

Calculated for C₁₈H₁₃N₅O, 0.75H₂O: C, 65.74%; H, 4.44%; N, 21.30%.Found: C, 65.58%; H, 4.50%; N, 21.05%.

Example 381 5-[3-(Biphenyl-4-yloxymethyl)phenyl]-1H-tetrazole

To a solution of 4-phenylphenol (10.0 g, 59 mmol) in dryN,N-dimethyl-formamide (45 mL) kept under an atmosphere of nitrogen,sodium hydride (2.82 g, 71 mmol, 60% dispersion in oil) was added inportions and the reaction mixture was stirred until gas evolutionceased. A solution of m-cyanobenzyl bromide (13 g, 65 mmol) in dryN,N-dimethylformamide (45 mL) was added dropwise and the reactionmixture was stirred at room temperature for 18 hours. The reactionmixture was poured on to ice water (150 mL). The precipitate wasfiltered of and washed with 50% ethanol (3×50 mL), ethanol (2×50 mL),diethyl ether (80 mL), and dried in vacuo at 50° C. for 18 hoursaffording crude 17.39 g of 3-(biphenyl-4-yloxymethyl)benzonitrile as asolid.

¹H NMR (200 MHz, CDCl₃) δ_(H) 5.14 (s, 2H), 7.05 (m, 2H), 7.30-7.78 (m,11H).

To a mixture of sodium azide (2.96 g, 45.6 mmol) and ammonium chloride(2.44 g, 45.6 mmol) in dry N,N-dimethylformamide (100 mL) under anatmosphere of nitrogen, 3-(biphenyl-4-yloxymethyl)-benzonitrile (10.0 g,35.0 mmol) was added and the reaction mixture was stirred at 125° C. for18 hours. The cooled reaction mixture was poured on to a mixture of 1Nhydrochloric acid (60 mL) and ice water (500 mL). The precipitate wasfiltered off and washed with water (3×100 mL), 50% ethanol (3×100 mL),ethanol (50 mL), diethyl ether (50 mL), ethanol (80 mL), and dried invacuo at 50° C. for 18 hours affording 8.02 g (70%) of the titlecompound.

¹H NMR (200 MHz, DMSO-d₆) δ_(H) 5.31 (s, 2H), 7.19 (m, 2H), 7.34 (m,1H), 7.47 (m, 2H), 7.69 (m, 6H), 8.05 (dt, 1H), 8.24 (s, 1H).

Example 382 5-(3-Phenoxymethyl)-phenyl)-tetrazole

3-Bromomethylbenzonitrile (5.00 g, 25.5 mmol) was dissolved inN,N-dimethylformamide (50 mL), phenol (2.40 g, 25.5 mmol) and potassiumcarbonate (10.6 g, 77 mmol) were added. The mixture was stirred at roomtemperature for 16 hours. The mixture was poured into water (400 mL) andextracted with ethyl acetate (2×200 mL). The combined organic extractswere washed with water (2×100 mL), dried (MgSO₄) and evaporated in vacuoto afford 5.19 g (97%) 3-(phenoxymethyl)benzonitrile as an oil.

TLC: R_(f)=0.38 (Ethyl acetate/heptane=1:4)

The above benzonitrile (5.19 g, 24.8 mmol) was dissolved inN,N-dimethylformamide (100 mL) and sodium azide (1.93 g, 30 mmol) andammonium chloride (1.59 g, 30 mmol) were added and the mixture washeated at 140° C. for 16 hours. After cooling, the mixture was pouredinto water (800 mL). The aqueous mixture was washed with ethyl acetate(200 mL). The pH of the aqueous phase was adjusted to 1 with 5 Nhydrochloric acid and stirred at room temperature for 30 minutes.Filtration, washing with water and drying in vacuo at 50° C. afforded2.06 g (33%) of the title compound as a solid.

¹H NMR (200 MHz, CDCl₃+DMSO-d₆) δ_(H) 5.05 (s, 2H), 6.88 (m, 3H), 7.21(m, 2H), 7.51 (m, 2H), 7.96 (dt, 1H), 8.14 (s, 1H).

Example 383 5-[3-(Biphenyl-4-ylmethoxy)phenyl]-1H-tetrazole

To a solution of 3-cyanophenol (5.0 g, 40.72 mmol) in dryN,N-dimethylformamide (100 mL) kept under an atmosphere of nitrogen,sodium hydride (2 g, 48.86 mmol, 60% dispersion in oil) was added inportions and the reaction mixture was stirred until gas evolutionceased. p-Phenylbenzyl chloride (9.26 g, 44.79 mmol) and potassiumiodide (0.2 g, 1.21 mmol) were added and the reaction mixture wasstirred at room temperature for 60 hours. The reaction mixture waspoured on to a mixture of saturated sodium carbonate (100 mL) and icewater (300 mL). The precipitate was filtered of and washed with water(3×100 mL), n-hexane (2×80 mL) and dried in vacuo at 50° C. for 18 hoursaffording 11.34 g (98%) of 3-(biphenyl-4-ylmethoxy)-benzonitrile as asolid.

To a mixture of sodium azide (2.37 g, 36.45 mmol) and ammonium chloride(1.95 g, 36.45 mmol) in dry N,N-dimethylformamide (100 mL) under anatmosphere of nitrogen, 3-(biphenyl-4-ylmethoxy)benzonitrile (8.0 g,28.04 mmol) was added and the reaction mixture was stirred at 125° C.for 18 hours. To the cooled reaction mixture water (100 mL) was addedand the reaction mixture stirred for 0.75 hour. The precipitate wasfiltered off and washed with water, 96% ethanol (2×50 mL), and dried invacuo at 50° C. for 18 hours affording 5.13 g (56%) of the titlecompound.

¹H NMR (200 MHz, DMSO-d₆) δ_(H) 5.29 (s, 2H), 7.31 (dd, 1H), 7.37-7.77(m, 12H).

Example 384 5-[4-(Biphenyl-4-ylmethoxy)-3-methoxyphenyl]-1H-tetrazol

This compound was made similarly as described in example 383.

Example 385

Example 386 5-(2-Naphtylmethyl)-1H-tetrazole

This compound was prepared similarly as described in example 379, step2.

Example 387 5-(1-Naphtylmethyl)-1H-tetrazole

This compound was prepared similarly as described in example 379, step2.

Example 388 5-[4-(Biphenyl-4-yloxymethyl)phenyl]-1H-tetrazole

A solution of alpha-bromo-p-tolunitrile (5.00 g, 25.5 mmol),4-phenylphenol (4.56 g, 26.8 mmol), and potassium carbonate (10.6 g,76.5 mmol) in N,N-dimethylformamide (75 mL) was stirred vigorously for16 hours at room temperature. Water (75 mL) was added and the mixturewas stirred at room temperature for 1 hour. The precipitate was filteredoff and washed with thoroughly with water. Drying in vacuo over night at50° C. afforded 7.09 g (97%) of 4-(biphenyl-4-yloxymethyl)benzonitrileas a solid.

The above benzonitrile (3.00 g, 10.5 mmol) was dissolved inN,N-dimethylformamide (50 mL), and sodium azide (1.03 g, 15.8 mmol) andammonium chloride (0.84 g, 15.8 mmol) were added and the mixture wasstirred 16 hours at 125° C. The mixture was cooled to room temperatureand water (50 mL) was added. The suspension was stirred overnight,filtered, washed with water and dried in vacuo at 50° C. for 3 days togive crude 3.07 g (89%) of the title compound. From the mother liquorcrystals were collected and washed with water, dried by suction to give0.18 g (5%) of the title compound as a solid.

¹H NMR (200 MHz, DMSO-d₆): δ_(H) 5.21 (s, 2H), 7.12 (d, 2H), 7.30 (t,1H), 7.42 (t, 2H), 7.56-7.63 (m, 6H), 8.03 (d, 2H).

Calculated for C₂₀H₁₆N₄O, 2H₂O: C, 65.92%; H, 5.53%; N, 15.37%. Found:C, 65.65%; H, 5.01%; N, 14.92%.

Example 389

This compound was prepared similarly as described in example 383.

Example 390

Example 391

Example 392

Example 393 5-(3-(Biphenyl-4-yloxymethyl)-benzyl)-1H-tetrazole

Example 394 5-(1-Naphthyl)-1H-tetrazole

This compound was prepared similarly as described in example 379, step2.

Example 3955-[3-Methoxy-4-(4-methylsulfonylbenzyloxy)phenyl]-1H-tetrazole

This compound was made similarly as described in example 383.

Example 396 5-(2-Naphthyl)-1H-tetrazole

This compound was prepared similarly as described in example 379, step2.

Example 397 2-Amino-N-(1H-tetrazol-5-yl)-benzamide

Example 398 5-(4-Hydroxy-3-methoxyphenyl)-1H-tetrazole

This compound was prepared similarly as described in example 379, step2.

Example 399 4-(2H-Tetrazol-5-ylmethoxy)benzoic acid

To a mixture of methyl 4-hydroxybenzoate (30.0 g, 0.20 mol), sodiumiodide (30.0 g, 0.20 mol) and potassium carbonate (27.6 g, 0.20 mol) inacetone (2000 mL) was added chloroacetonitrile (14.9 g, 0.20 mol). Themixture was stirred at RT for 3 days. Water was added and the mixturewas acidified with 1N hydrochloric acid and the mixture was extractedwith diethyl ether. The combined organic layers were dried over Na₂SO₄and concentrated in vacuo. The residue was dissolved in acetone andchloroacetonitrile (6.04 g, 0.08 mol), sodium iodide (12.0 g, 0.08 mol)and potassium carbonate (11.1 g, 0.08 mol) were added and the mixturewas stirred for 16 hours at RT and at 60° C. More chloroacetonitrile wasadded until the conversion was 97%. Water was added and the mixture wasacidified with 1N hydrochloric acid and the mixture was extracted withdiethyl ether. The combined organic layers were dried over Na₂SO₄ andconcentrated in vacuo to afford methyl 4-cyanomethyloxybenzoate inquantitative yield. This compound was used without further purificationin the following step.

A mixture of methyl 4-cyanomethyloxybenzoate (53.5 g, 0.20 mol), sodiumazide (16.9 g, 0.26 mol) and ammonium chloride (13.9 g, 0.26 mol) in DMF1000 (mL) was refluxed overnight under N₂. After cooling, the mixturewas concentrated in vacuo. The residue was suspended in cold water andextracted with ethyl acetate. The combined organic phases were washedwith brine, dried over Na₂SO₄ and concentrated in vacuo, to affordmethyl 4-(2H-tetrazol-5-ylmethoxy)benzoate. This compound was used assuch in the following step.

Methyl 4-(2H-Tetrazol-5-ylmethoxy)-benzoate was refluxed in 3N sodiumhydroxide. The reaction was followed by TLC (DCM:MeOH=9:1). The reactionmixture was cooled, acidified and the product filtered off. The impureproduct was washed with DCM, dissolved in MeOH, filtered and purified bycolumn chromatography on silica gel (DCM:MeOH=9:1). The resultingproduct was recrystallised from DCM:MeOH=95:5. This was repeated untilthe product was pure. This afforded 13.82 g (30%) of the title compound.

¹H-NMR (DMSO-d₆): 4.70 (2H, s), 7.48 (2H, d), 7.73 (2H, d), 13 (1H, bs).

Example 400 4-(2H-Tetrazol-5-ylmethylsulfanyl)benzoic acid

To a solution of sodium hydroxide (10.4 g, 0.26 mol) in degassed water(600 mL) was added 4-mercaptobenzoic acid (20.0 g, 0.13 mol). Thissolution was stirred for 30 minutes. To a solution of potassiumcarbonate (9.0 g, 65 mmol) in degassed water (400 mL) was addedchloroacetonitrile (9.8 g, (0.13 mol) portion-wise. These two solutionswere mixed and stirred for 48 hours at RT under N₂. The mixture wasfiltered and washed with heptane. The aqueous phase was acidified with3N hydrochloric acid and the product was filtered off, washed with waterand dried, affording 4-cyanomethylsulfanylbenzoic acid (27.2 g, 88%).This compound was used without further purification in the followingstep.

A mixture of 4-cyanomethylsulfanylbenzoic acid (27.2 g, 0.14 mol),sodium azide (11.8 g, 0.18 mol) and ammonium chloride (9.7 g, 0.18 mol)in DMF (1000 mL) was refluxed overnight under N₂. The mixture wasconcentrated in vacuo. The residue was suspended in cold water andextracted with diethyl ether. The combined organic phases were washedwith brine, dried over Na₂SO₄ and concentrated in vacuo. Water was addedand the precipitate was filtered off. The aqueous layer was concentratedin vacuo, water was added and the precipitate filtered off. The combinedimpure products were purified by column chromatography usingDCM:MeOH=9:1 as eluent, affording the title compound (5.2 g, 16%).

¹H-NMR (DMSO-d₆): 5.58 (2H, s), 7.15 (2H, d), 7.93 (2H, d), 12.7 (1H,bs).

Example 401 3-(2H-Tetrazol-5-yl)-9H-carbazole

3-Bromo-9H-carbazole was prepared as described by Smith et al. inTetrahedron 1992, 48, 7479-7488.

A solution of 3-bromo-9H-carbazole (23.08 g, 0.094 mol) and cuprouscyanide (9.33 g, 0.103 mol) in N-methyl-pyrrolidone (300 ml) was heatedat 200° C. for 5 h. The cooled reaction mixture was poured on to water(600 ml) and the precipitate was filtered off and washed with ethylacetate (3×50 ml). The filtrate was extracted with ethyl acetate (3×250ml) and the combined ethyl acetate extracts were washed with water (150ml), brine (150 ml), dried (MgSO₄) and concentrated in vacuo. Theresidue was crystallised from heptanes and recrystallised fromacetonitrile (70 ml) affording 7.16 g (40%) of 3-cyano-9H-carbazole as asolid.

M.p. 180-181° C.

3-Cyano-9H-carbazole (5.77 g, 30 mmol) was dissolved inN,N-dimethylformamide (150 ml), and sodium azide (9.85 g, 152 mmol),ammonium chloride (8.04 g, 150 mmol) and lithium chloride (1.93 g, 46mmol) were added and the mixture was stirred for 20 h at 125° C. To thereaction mixture was added an additional portion of sodium azide (9.85g, 152 mmol) and ammonium chloride (8.04 g, 150 mmol) and the reactionmixture was stirred for an additional 24 h at 125° C. The cooledreaction mixture was poured on to water (500 ml). The suspension wasstirred for 0.5 h, and the precipitate was filtered off and washed withwater (3×200 ml) and dried in vacuo at 50° C. The dried crude productwas suspended in diethyl ether (500 ml) and stirred for 2 h, filteredoff and washed with diethyl ether (2×200 ml) and dried in vacuo at 50°C. affording 5.79 g (82%) of the title compound as a solid.

¹H-NMR (DMSO-d₆): δ 11.78 (1H, bs), 8.93 (1H, d), 8.23 (1H, d), 8.14(1H, dd), 7.72 (1H, d), 7.60 (1H, d), 7.49 (1H, t), 7.28 (1H, t);HPLC-MS (Method C): m/z: 236 (M+1); Rt=2.77 min.

The following commercially available tetrazoles do all bind to the HisB10 Zn²⁺ site of the insulin hexamer:

Example 402 5-(3-Tolyl)-1H-tetrazole

Example 403 5-(2-Bromophenyl)tetrazole

Example 404 5-(4-Ethoxalylamino-3-nitrophenyl)tetrazole

Example 405

Example 406

Example 407

Example 408

Example 409 Tetrazole

Example 410 5-Methyltetrazole

Example 411 5-Benzyl-2H-tetrazole

Example 412 4-(2H-Tetrazol-5-yl)benzoic acid

Example 413 5-Phenyl-2H-tetrazole

Example 414 5-(4-Chlorophenylsulfanylmethyl)-2H-tetrazole

Example 415 5-(3-Benzyloxyphenyl)-2H-tetrazole

Example 416 2-Phenyl-6-(1H-tetrazol-5-yl)-chromen-4-one

Example 417

Example 418

Example 419

Example 420

Example 421

Example 422 5-(4-Bromo-phenyl)-1H-tetrazole

Example 423

Example 424

Example 425

Example 426

Example 427

Example 428

Example 429

Example 430

Example 431

Example 432

Example 433

Example 434

Example 435

Example 436

Example 437

Example 438

Example 439

Example 440

Example 441

Example 442

Example 443

Example 444

Example 445

Example 446

Example 447

Example 448

Example 449

Example 450

Example 451

Example 452

General Procedure (H) for Preparation of Compounds of General FormulaI₇:

wherein A¹, AR¹, and AR² are as defined above.

The reaction is generally known as a reductive alkylation reaction andis generally performed by stirring an aldehyde with an amine at low pH(by addition of an acid, such as acetic acid or formic acid) in asolvent such as THF, DMF, NMP, methanol, ethanol, DMSO, dichloromethane,1,2-dichloroethane, trimethyl orthoformate, triethyl orthoformate, or amixture of two or more of these. As reducing agent sodium cyanoborohydride or sodium triacetoxy borohydride may be used. The reactionis performed between 20° C. and 120° C., preferably at room temperature.

When the reductive alkylation is complete, the product is isolated byextraction, filtration, chromatography or other methods known to thoseskilled in the art.

The general procedure (H) is further illustrated in the followingexample 453:

Example 453 General Procedure (H)Biphenyl-4-ylmethyl-[3-(2H-tetrazol-5-yl)phenyl]amine

A solution of 5-(3-aminophenyl)-2H-tetrazole (example 589, 48 mg, 0.3mmol) in DMF (250 μL) was mixed with a solution of4-biphenylylcarbaldehyde (54 mg, 0.3 mmol) in DMF (250 μL) and aceticacid glacial (250 μL) was added to the mixture followed by a solution ofsodium cyano borohydride (15 mg, 0.24 mmol) in methanol (250 μL). Theresulting mixture was shaken at room temperature for 2 hours. Water (2mL) was added to the mixture and the resulting mixture was shaken atroom temperature for 16 hours. The mixture was centrifugated (6000 rpm,10 minutes) and the supernatant was removed by a pipette. The residuewas washed with water (3 mL), centrifugated (6000 rpm, 10 minutes) andthe supernatant was removed by a pipette. The residue was dried in vacuoat 40° C. for 16 hours to afford the title compound as a solid.

HPLC-MS (Method C): m/z: 328 (M+1), 350 (M+23); Rt=4.09 min.

Example 454 General Procedure (H)Benzyl-[3-(2H-tetrazol-5-yl)phenyl]amine

HPLC-MS (Method D): m/z: 252 (M+1); Rt=3.74 min.

Example 455 General Procedure (H)(4-Methoxybenzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine

HPLC-MS (Method D): m/z: 282.2 (M+1); Rt=3.57 min.

Example 456 General Procedure (H)4-{[3-(2H-Tetrazol-5-yl)phenylamino]methyl}phenol

HPLC-MS (Method D): m/z: 268.4 (M+1); Rt=2.64 min.

Example 457 General Procedure (H)(4-Nitrobenzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine

HPLC-MS (Method D): m/z: 297.4 (M+1); Rt=3.94 min.

Example 458 General Procedure (H)(4-Chlorobenzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine

HPLC-MS (Method D): m/z: 287.2 (M+1); Rt=4.30 min.

Example 459 General Procedure (H)(2-Chlorobenzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine

HPLC-MS (Method D): m/z: 286 (M+1); Rt=4.40 min.

Example 460 General Procedure (H)(4-Bromobenzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine

HPLC-MS (Method D): m/z: 332 (M+1); Rt=4.50 min.

Example 461 General Procedure (H)(3-Benzyloxybenzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine

HPLC-MS (Method D): m/z: 358 (M+1); Rt=4.94 min.

Example 462 General Procedure (H)Naphthalen-1-ylmethyl-[3-(2H-tetrazol-5-yl)phenyl]amine

HPLC-MS (Method D): m/z: 302 (M+1); Rt=4.70 min.

Example 463 General Procedure (H)Naphthalen-2-ylmethyl-[3-(2H-tetrazol-5-yl)phenyl]amine

HPLC-MS (Method D): m/z: 302 (M+1); Rt=4.60 min.

Example 464 General Procedure (H)4-{[3-(2H-Tetrazol-5-yl)phenylamino]methyl}benzoic acid

HPLC-MS (Method D): m/z: 296 (M+1); Rt=3.24 min.

Example 465 General Procedure (H)[3-(2H-Tetrazol-5-yl)-phenyl]-[3-(3-trifluoromethyl-phenoxy)benzyl]amine

HPLC-MS (Method D): m/z: 412 (M+1); Rt=5.54 min.

Example 466 General Procedure (H)(3-Phenoxybenzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine

HPLC-MS (Method D): m/z: 344 (M+1); Rt=5.04 min.

Example 467 General Procedure (H)(4-Phenoxy-benzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine

HPLC-MS (Method D): m/z: 344 (M+1); Rt=5.00 min.

Example 468 General Procedure (H)(4-{[3-(2H-Tetrazol-5-yl)phenylamino]methyl}phenoxy)acetic acid

HPLC-MS (Method D): m/z: 326 (M+1); Rt=3.10 min.

Example 469 General Procedure (H)(4-Benzyloxybenzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine

HPLC-MS (Method D): m/z: 358 (M+1); Rt=4.97 min.

Example 470 General Procedure (H)3-(4-{[3-(2H-Tetrazol-5-yl)phenylamino]methyl}phenyl)acrylic acid

HPLC-MS (Method D): m/z: 322 (M+1); Rt=3.60 min.

Example 471 General Procedure (H)Dimethyl-(4-{[3-(2H-tetrazol-5-yl)phenylamino]methyl}naphthalen-1-yl)amine

HPLC-MS (Method D): m/z: 345 (M+1); Rt=3.07 min.

Example 472 General Procedure (H)(4′-Methoxybiphenyl-4-ylmethyl)-[3-(2H-tetrazol-5-yl)phenyl]amine

HPLC-MS (Method D): m/z: 358 (M+1); Rt=4.97 min.

Example 473 General Procedure (H)(2′-Chlorobiphenyl-4-ylmethyl)-[3-(2H-tetrazol-5-yl)phenyl]amine

HPLC-MS (Method D): m/z: 362 (M+1); Rt=5.27 min.

Example 474 General Procedure (H)Benzyl-[4-(2H-tetrazol-5-yl)phenyl]amine

For preparation of starting material, see example 590.

HPLC-MS (Method D): m/z: 252 (M+1); Rt=3.97 min.

Example 475 General Procedure (H)(4-Methoxybenzyl)-[4-(2H-tetrazol-5-yl)phenyl]amine

HPLC-MS (Method D): m/z: 282 (M+1); Rt=3.94 min.

Example 476 General Procedure (H)4-{[4-(2H-Tetrazol-5-yl)phenylamino]methyl}phenol

HPLC-MS (Method D): m/z: 268 (M+1); Rt=3.14 min.

Example 477 General Procedure (H)(4-Nitrobenzyl)-[4-(2H-tetrazol-5-yl)phenyl]amine

HPLC-MS (Method D): m/z: (M+1); Rt=3.94 min.

Example 478 General Procedure (H)(4-Chlorobenzyl)-[4-(2H-tetrazol-5-yl)phenyl]amine

HPLC-MS (Method D): m/z: (M+1); Rt=4.47 min.

Example 479 General Procedure (H)(2-Chlorobenzyl)-[4-(2H-tetrazol-5-yl)phenyl]amine

HPLC-MS (Method D): m/z: 286 (M+1); Rt=4.37 min.

Example 480 General Procedure (H)(4-Bromobenzyl)-[4-(2H-tetrazol-5-yl)phenyl]amine

HPLC-MS (Method D): m/z: 331 (M+1); Rt=4.57 min.

Example 481 General Procedure (H)(3-Benzyloxybenzyl)-[4-(2H-tetrazol-5-yl)phenyl]amine

HPLC-MS (Method D): m/z: 358 (M+1); Rt=5.07 min.

Example 482 General Procedure (H)Naphthalen-1-ylmethyl-[4-(2H-tetrazol-5-yl)phenyl]amine

HPLC-MS (Method D): m/z: 302 (M+1); Rt=4.70 min.

Example 483 General Procedure (H)Naphthalen-2-ylmethyl-[4-(2H-tetrazol-5-yl)phenyl]amine

HPLC-MS (Method D): m/z: 302 (M+1); Rt=4.70 min.

Example 484 General Procedure (H)Biphenyl-4-ylmethyl-[4-(2H-tetrazol-5-yl)phenyl]amine

HPLC-MS (Method D): m/z: 328 (M+1); Rt=5.07 min.

Example 485 General Procedure (H)4-{[4-(2H-Tetrazol-5-yl)phenylamino]methyl}benzoic acid

HPLC-MS (Method D): m/z: 296 (M+1); Rt=3.34 min.

Example 486 General Procedure (H)[4-(2H-Tetrazol-5-yl)phenyl]-[3-(3-trifluoromethylphenoxy)benzyl]amine

HPLC-MS (Method D): m/z: 412 (M+1); Rt=5.54 min.

Example 487 General Procedure (H)(3-Phenoxybenzyl)-[4-(2H-tetrazol-5-yl)phenyl]amine

HPLC-MS (Method D): m/z: 344 (M+1); Rt=5.07 min.

Example 488 General Procedure (H)(4-Phenoxybenzyl)-[4-(2H-tetrazol-5-yl)-phenyl]-amine

HPLC-MS (Method D): m/z: 344 (M+1); Rt=5.03 min.

Example 489 General Procedure (H)3-{[4-(2H-Tetrazol-5-yl)phenylamino]methyl}benzoic acid

HPLC-MS (Method D): m/z: 286 (M+1); Rt=3.47 min.

Example 490 General Procedure (H)(4-{[4-(2H-Tetrazol-5-yl)phenylamino]methyl}phenoxy)acetic acid

HPLC-MS (Method D): m/z: 326 (M+1); Rt=3.40 min.

Example 491 General Procedure (H)(4-Benzyloxybenzyl)-[4-(2H-tetrazol-5-yl)phenyl]amine

HPLC-MS (Method D): m/z: 358 (M+1); Rt=5.14 min.

Example 492 General Procedure (H)3-(4-{[4-(2H-Tetrazol-5-yl)phenylamino]methyl}phenyl)acrylic acid

HPLC-MS (Method D): m/z: 322 (M+1); Rt=3.66 min.

Example 493 General Procedure (H)Dimethyl-(4-{[4-(2H-tetrazol-5-yl)phenylamino]methyl}naphthalen-1-yl)amine

HPLC-MS (Method D): m/z: 345 (M+1); Rt=3.10 min.

Example 494 General Procedure (H)(4′-Methoxybiphenyl-4-ylmethyl)-[4-(2H-tetrazol-5-yl)phenyl]amine

HPLC-MS (Method D): m/z: 358 (M+1); Rt=5.04 min.

Example 495 General Procedure (H)(2′-Chlorobiphenyl-4-ylmethyl)-[4-(2H-tetrazol-5-yl)-phenyl]-amine

HPLC-MS (Method D): m/z: 362 (M+1); Rt=5.30 min.

General Procedure (I) for Preparation of Compounds of General FormulaI₈:

wherein A¹, AR¹, and AR² are as defined above.

This procedure is very similar to general procedure (A), the onlydifference being the carboxylic acid is containing a tetrazole moiety.When the acylation is complete, the product is isolated by extraction,filtration, chromatography or other methods known to those skilled inthe art.

The general procedure (I) is further illustrated in the followingexample 496:

Example 496 General Procedure (I)4-[4-(2H-Tetrazol-5-yl)benzoylamino]benzoic acid

To a solution of 4-(2H-tetrazol-5-yl)benzoic acid (example 412, 4 mmol)and HOAt (4.2 mmol) in DMF (6 mL) was added1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (4.2 mmol)and the resulting mixture was stirred at room temperature for 1 hour. Analiquot of this HOAt-ester solution (0.45 mL) was mixed with 0.25 mL ofa solution of 4-aminobenzoic acid (1.2 mmol in 1 mL DMF). (Anilines ashydrochlorides can also be utilised, a slight excess of triethylaminewas added to the hydrochloride suspension in DMF prior to mixing withthe HOAt-ester.) The resulting mixture was shaken for 3 days at roomtemperature. 1N hydrochloric acid (2 mL) was added and the mixture wasshaken for 16 hours at room temperature. The solid was isolated bycentrifugation (alternatively by filtration or extraction) and waswashed with water (3 mL). Drying in vacuo at 40° C. for 2 days affordedthe title compound.

HPLC-MS (Method D): m/z: 310 (M+1); Rt=2.83 min.

Example 497 General Procedure (I)3-[4-(2H-Tetrazol-5-yl)benzoylamino]benzoic acid

HPLC-MS (Method D): m/z: 310 (M+1); Rt=2.89 min.

Example 498 General Procedure (I)3-{4-[4-(2H-Tetrazol-5-yl)benzoylamino]phenyl}acrylic acid

HPLC-MS (Method D): m/z: 336 (M+1); Rt=3.10 min.

Example 499 General Procedure (I)3-{4-[4-(2H-Tetrazol-5-yl)benzoylamino]phenyl}propionic acid

HPLC-MS (Method D): m/z: 338 (M+1); Rt=2.97 min.

Example 500 General Procedure (I)3-Methoxy-4-[4-(2H-tetrazol-5-yl)benzoylamino]benzoic acid

HPLC-MS (Method D): m/z: 340 (M+1); Rt=3.03 min.

Example 501 General Procedure (I)N-(4-Benzyloxyphenyl)-4-(2H-tetrazol-5-yl)benzamide

HPLC-MS (Method D): m/z: 372 (M+1); Rt=4.47 min.

Example 502 General Procedure (I)N-(4-Phenoxyphenyl)-4-(2H-tetrazol-5-yl)benzamide

HPLC-MS (Method D): m/z: 358 (M+1); Rt=4.50 min.

Example 503 General Procedure (I)N-(9H-Fluoren-2-yl)-4-(2H-tetrazol-5-yl)benzamide

HPLC-MS (Method D): m/z: 354 (M+1); Rt=4.60 min.

Example 504 General Procedure (I)N-(9-Ethyl-9H-carbazol-2-yl)-4-(2H-tetrazol-5-yl)benzamide

HPLC-MS (Method D): m/z: 383 (M+1); Rt=4.60 min.

Example 505 General Procedure (I) N-Phenyl-4-(2H-tetrazol-5-yl)benzamide

HPLC-MS (Method D): m/z: 266 (M+1); Rt=3.23 min.

Example 506 General Procedure (I)4-[4-(2H-Tetrazol-5-ylmethoxy)benzoylamino]benzoic acid

The starting material was prepared as described in example 399.

HPLC-MS (Method D): m/z: 340 (M+1); Rt=2.83 min.

Example 507 General Procedure (I)3-[4-(2H-Tetrazol-5-ylmethoxy)benzoylamino]benzoic acid

HPLC-MS (Method D): m/z: 340 (M+1); Rt=2.90 min.

Example 508 General Procedure (I)3-{4-[4-(2H-Tetrazol-5-ylmethoxy)benzoylamino]phenyl}acrylic acid

HPLC-MS (Method D): m/z: 366 (M+1); Rt=3.07 min.

Example 509 General Procedure (I)3-{4-[4-(2H-Tetrazol-5-ylmethoxy)benzoylamino]phenyl}propionic acid

HPLC-MS (Method D): m/z: 368 (M+1); Rt=2.97 min.

Example 510 General Procedure (I)3-Methoxy-4-[4-(2H-tetrazol-5-ylmethoxy)benzoylamino]benzoic acid

HPLC-MS (Method D): m/z: 370 (M+1); Rt=3.07 min.

Example 511 General Procedure (I)N-(4-Benzyloxyphenyl)-4-(2H-tetrazol-5-ylmethoxy)benzamide

HPLC-MS (Method D): m/z: 402 (M+1); Rt=4.43 min.

Example 512 General Procedure (I)N-(4-Phenoxyphenyl)-4-(2H-tetrazol-5-ylmethoxy)benzamide

HPLC-MS (Method D): m/z: 388 (M+1); Rt=4.50 min.

Example 513 General Procedure (I)N-(9H-Fluoren-2-yl)-4-(2H-tetrazol-5-ylmethoxy)benzamide

HPLC-MS (Method D): m/z: 384 (M+1); Rt=4.57 min.

Example 514 General Procedure (I)N-(9-Ethyl-9H-carbazol-2-yl)-4-(2H-tetrazol-5-ylmethoxy)benzamide

HPLC-MS (Method D): m/z: 413 (M+1); Rt=4.57 min.

Example 515 General Procedure (I)N-Phenyl-4-(2H-tetrazol-5-ylmethoxy)benzamide

HPLC-MS (Method D): m/z: 296 (M+1); Rt=3.23 min.

Example 516 General Procedure (I)4-[4-(2H-Tetrazol-5-ylmethylsulfanyl)benzoylamino]benzoic acid

The starting material was prepared as described in example 400.

HPLC-MS (Method D): m/z: 356 (M+1); Rt=2.93 min.

Example 517 General Procedure (I)3-[4-(2H-Tetrazol-5-ylmethylsulfanyl)benzoylamino]benzoic acid

HPLC-MS (Method D): m/z: 356 (M+1); Rt=3.00 min.

Example 518 General Procedure (I)3-{4-[4-(2H-Tetrazol-5-ylmethylsulfanyl)benzoylamino]phenyl}acrylic acid

HPLC-MS (Method D): m/z: 382 (M+1); Rt=3.26 min.

Example 519 General Procedure (I)3-{4-[4-(2H-Tetrazol-5-ylmethylsulfanyl)benzoylamino]phenyl}propionicacid

HPLC-MS (Method D): m/z: 384 (M+1); Rt=3.10 min.

Example 520 General Procedure (I)3-Methoxy-4-[4-(2H-tetrazol-5-ylmethylsulfanyl)benzoylamino]benzoic acid

HPLC-MS (Method D): m/z: 386 (M+1); Rt=3.20 min.

Example 521 General Procedure (I)N-(4-Benzyloxyphenyl)-4-(2H-tetrazol-5-ylmethylsulfanyl)benzamide

HPLC-MS (Method D): m/z: 418 (M+1); Rt=4.57 min.

Example 522 General Procedure (I)N-(4-Phenoxyphenyl)-4-(2H-tetrazol-5-ylmethylsulfanyl)benzamide

HPLC-MS (Method D): m/z: 404 (M+1); Rt=4.60 min.

Example 523 General Procedure (I)N-(9H-Fluoren-2-yl)-4-(2H-tetrazol-5-ylmethylsulfanyl)benzamide

HPLC-MS (Method D): m/z: 400 (M+1); Rt=4.67 min.

Example 524 General Procedure (I)N-(9-Ethyl-9H-carbazol-2-yl)-4-(2H-tetrazol-5-ylmethylsulfanyl)benzamide

HPLC-MS (Method D): m/z: 429 (M+1); Rt=4.67 min.

Example 525 General Procedure (I)N-Phenyl-4-(2H-tetrazol-5-ylmethylsulfanyl)benzamide

HPLC-MS (Method D): m/z: 312 (M+1); Rt=3.40 min.

General Procedure (J) for Solution Phase Preparation of Amides ofGeneral Formula I₉:

wherein AR2 is as defined above.

This general procedure (J) is further illustrated in the followingexample.

Example 526 General Procedure (J)9-(3-Chlorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

3-(2H-Tetrazol-5-yl)-9H-carbazole (example 401, 17 g, 72.26 mmol) wasdissolved in N,N-dimethylformamide (150 mL). Triphenylmethyl chloride(21.153 g, 75.88 mmol) and triethylamine (20.14 mL, 14.62 g, 144.50mmol) were added consecutively. The reaction mixture was stirred for 18hours at room temperature, poured into water (1.5 L) and stirred for anadditional 1 hour. The crude product was filtered off and dissolved indichloromethane (500 mL). The organic phase was washed with water (2×250mL) and dried with magnesium sulfate (1 h). Filtration followed byconcentration yielded a solid which was triturated in heptanes (200 mL).Filtration furnished3-[2-(triphenylmethyl)-2H-tetrazol-5-yl]-9H-carbazole (31.5 g) which wasused without further purification.

¹H-NMR (CDCl₃): δ 8.87 (1H, d), 8.28 (1H, bs), 8.22 (1H, dd), 8.13 (1H,d), 7.49 (1H, d), 7.47-7.19 (18H, m); HPLC-MS (Method C): m/z: 243(triphenylmethyl); Rt=5.72 min.

3-[2-(Triphenylmethyl)-2H-tetrazol-5-yl]-9H-carbazole (200 mg, 0.42mmol) was dissolved in methyl sulfoxide (1.5 mL). Sodium hydride (34 mg,60%, 0.85 mmol) was added, and the resulting suspension was stirred for30 min at room temperature. 3-Chlorobenzyl chloride (85 μL, 108 mg, 0.67mmol) was added, and the stirring was continued at 40° C. for 18 hours.The reaction mixture was cooled to ambient temperature and poured into0.1 N hydrochloric acid (aq.) (15 mL) The precipitated solid wasfiltered off and washed with water (3×10 mL) to furnish9-(3-chlorobenzyl)-3-[2-(triphenylmethyl)-2H-tetrazol-5-yl]-9H-carbazole,which was dissolved in a mixture of tetrahydrofuran and 6 N hydrochloricacid (aq.) (9:1) (10 mL) and stirred at room temperature for 18 hours.The reaction mixture was poured into water (100 mL). The solid wasfiltered off and rinsed with water (3×10 mL) and dichloromethane (3×10mL) to yield the title compound (127 mg). No further purification wasnecessary.

¹H-NMR (DMSO-d₆): δ 8.89 (1H, d), 8.29 (1H, d), 8.12 (1H, dd), 7.90 (1H,d), 7.72 (1H, d), 7.53 (1H, t), 7.36-7.27 (4H, m), 7.08 (1H, bt), 5.78(2H, s); HPLC-MS (Method B): m/z: 360 (M+1); Rt=5.07 min.

The compounds in the following examples were prepared in a similarfashion. Optionally, the compounds can be further purified byrecrystallization from e.g. aqueous sodium hydroxide (1 N) or bychromatography.

Example 527 General Procedure (J)9-(4-Chlorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

HPLC-MS (Method C): m/z: 360 (M+1); Rt=4.31 min.

Example 528 General Procedure (J)9-(4-Methylbenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

HPLC-MS (Method C): m/z: 340 (M+1); Rt=4.26 min.

Example 529 General Procedure (J)3-(2H-Tetrazol-5-yl)-9-(4-trifluoromethylbenzyl)-9H-carbazole

HPLC-MS (Method C): m/z: 394 (M+1); Rt=4.40 min.

Example 530 General Procedure (J)9-(4-Benzyloxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

HPLC-MS (Method C): m/z: 432 (M+1); Rt=4.70 min.

Example 531 General Procedure (J)9-(3-Methylbenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

HPLC-MS (Method C): m/z: 340 (M+1); Rt=4.25 min.

Example 532 General Procedure (J)9-Benzyl-3-(2H-tetrazol-5-yl)-9H-carbazole

¹H-NMR (DMSO-d₆): δ 8.91 (1H, dd), 8.30 (1H, d), 8.13 (1H, dd), 7.90(1H, d), 7.73 (1H, d), 7.53 (1H, t), 7.36-7.20 (6H, m), 5.77 (2H, s).

Example 533 General Procedure (J)9-(4-Phenylbenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

¹H-NMR (DMSO-d₆): δ 8.94 (1H, s), 8.33 (1H, d), 8.17 (1H, dd), 7.95 (1H,d), 7.77 (1H, d), 7.61-7.27 (11H, m), 5.82 (2H, s).

Example 534 General Procedure (J)9-(3-Methoxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

HPLC-MS (Method C): m/z: 356 (M+1); Rt=3.99 min.

Example 535 General Procedure (J)9-(Naphthalen-2-ylmethyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

HPLC-MS (Method C): m/z: 376 (M+1); Rt=4.48 min.

Example 536 General Procedure (J)9-(3-Bromobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

HPLC-MS (Method C): m/z: 404 (M+1); Rt=4.33 min.

Example 537 General Procedure (J)9-(Biphenyl-2-ylmethyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

HPLC-MS (Method C): m/z: 402 (M+1); Rt=480 min.

Example 538 General Procedure (J)3-(2H-Tetrazol-5-yl)-9-[4-(1,2,3-thiadiazol-4-yl)benzyl]-9H-carbazole

Example 539 General Procedure (J)9-(2′-Cyanobiphenyl-4-ylmethyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

¹H-NMR (DMSO-d₆): δ 8.91 (1H, d), 8.31 (1H, d), 8.13 (1H, dd), 7.95 (1H,d), 7.92 (1H, d), 7.78 (1H, d), 7.75 (1H, dt), 7.60-7.47 (5H, m),7.38-7.28 (3H, m), 5.86 (2H, s); HPLC-MS (Method C): m/z: 427 (M+1);Rt=4.38 min.

Example 540 General Procedure (J)9-(4-Iodobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

HPLC-MS (Method C): m/z: 452 (M+1); Rt=4.37 min.

Example 541 General Procedure (J)9-(3,5-Bis(trifluoromethyl)benzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

HPLC-MS (Method C): m/z: 462 (M+1); Rt=4.70 min.

Example 542 General Procedure (J)9-(4-Bromobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

¹H-NMR (DMSO-d₆): δ 8.89 (1H, d), 8.29 (1H, d), 8.11 (1H, dd), 7.88 (1H,d), 7.70 (1H, d), 7.52 (1H, t), 7.49 (2H, d), 7.31 (1H, t), 7.14 (2H,d), 5.74 (2H, s); HPLC-MS (Method C): m/z: 404 (M+1); Rt=4.40 min.

Example 543 General Procedure (J)9-(Anthracen-9-ylmethyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

HPLC-MS (Method C): m/z: 426 (M+1); Rt=4.78 min.

Example 544 General Procedure (J)9-(4-Carboxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

3.6 fold excess sodium hydride was used.

¹H-NMR (DMSO-d₆): δ 12.89 (1H, bs), 8.89 (1H, d), 8.30 (1H, d), 8.10(1H, dd), 7.87 (1H, d), 7.86 (2H, d), 7.68 (1H, d), 7.51 (1H, t), 7.32(1H, t), 7.27 (2H, d), 5.84 (2H, s); HPLC-MS (Method C): m/z: 370 (M+1);Rt=3.37 min.

Example 545 General Procedure (J)9-(2-Chlorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

HPLC-MS (Method B): m/z: 360 (M+1); Rt=5.30 min.

Example 546 General Procedure (J)9-(4-Fluorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

¹H-NMR (DMSO-d₆): δ 8.88 (1H, d), 8.28 (1H, d), 8.10 (1H, dd), 7.89 (1H,d), 7.72 (1H, d), 7.52 (1H, t), 7.31 (1H, t), 7.31-7.08 (4H, m), 5.74(2H, s); HPLC-MS (Method C): m/z: 344 (M+1); Rt=4.10 min.

Example 547 General Procedure (J)9-(3-Fluorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

¹H-NMR (DMSO-d₆): δ 8.89 (1H, d), 8.29 (1H, d), 8.12 (1H, dd), 7.90 (1H,d), 7.72 (1H, d), 7.53 (1H, t), 7.37-7.27 (2H, m), 7.12-7.02 (2H, m),6.97 (1H, d), 5.78 (2H, s); HPLC-MS (Method C): m/z: 344 (M+1); Rt=4.10min.

Example 548 General Procedure (J)9-(2-Iodobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

HPLC-MS (Method C): m/z: 452 (M+1); Rt=4.58 min.

Example 549 General Procedure (J)9-(3-Carboxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

3.6 fold excess sodium hydride was used.

¹H-NMR (DMSO-d₆): δ 12.97 (1H, bs), 8.90 (1H, bs), 8.30 (1H, d), 8.12(1H, bd), 7.8 (1H, d), 7.82 (1H, m), 7.77 (1H, bs), 7.71 (1H, d), 7.53(1H, t), 7.46-7.41 (2H, m), 7.32 (1H, t), 5.84 (2H, s); HPLC-MS (MethodC): m/z: 370 (M+1); Rt=3.35 min.

Example 550 General Procedure (J)9-[4-(2-Propyl)benzyl]-3-(2H-tetrazol-5-yl)-9H-carbazole

¹H-NMR (DMSO-d₆): δ 8.87 (1H, d), 8.27 (1H, d), 8.10 (1H, dd), 7.87 (1H,d), 7.71 (1H, d), 7.51 (1H, t), 7.31 (1H, t), 7.15 (2H, d), 7.12 (2H,d), 5.69 (2H, s), 2.80 (1H, sept), 1.12 (6H, d); HPLC-MS (Method C):m/z: 368 (M+1); Rt=4.73 min.

Example 551 General Procedure (J)9-(3,5-Dimethoxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

HPLC-MS (Method C): m/z: 386 (M+1); Rt=4.03 min.

Example 552 General Procedure (J)3-(2H-Tetrazol-5-yl)-9-(2,4,5-trifluorobenzyl)-9H-carbazole

HPLC-MS (Method B): m/z: 380 (M+1); Rt=5.00 min.

Example 553 General Procedure (J)N-Methyl-N-phenyl-2-[3-(2H-tetrazol-5-yl)carbazol-9-yl]acetamide

HPLC-MS (Method B): m/z: 383 (M+1); Rt=4.30 min.

Example 554 General Procedure (J)9-(4-Methoxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

¹H-NMR (DMSO-d₆): δ 8.86 (1H, d), 8.26 (1H, d), 8.10 (1H, dd), 7.90 (1H,d), 7.73 (1H, d), 7.51 (1H, t), 7.30 (1H, t), 7.18 (2H, d), 6.84 (2H,d), 5.66 (2H, s), 3.67 (3H, s); HPLC-MS (Method B): m/z: 356 (M+1);Rt=4.73 min.

Example 555 General Procedure (J)9-(2-Methoxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

¹H-NMR (DMSO-d₆): δ 8.87 (1H, d), 8.27 (1H, d), 8.09 (1H, dd), 7.77 (1H,d), 7.60 (1H, d), 7.49 (1H, t), 7.29 (1H, t), 7.23 (1H, bt), 7.07 (1H,bd), 6.74 (1H, bt), 6.61 (1H, bd), 5.65 (2H, s), 3.88 (3H, s); HPLC-MS(Method B): m/z: 356 (M+1); Rt=4.97 min.

Example 556 General Procedure (J)9-(4-Cyanobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

HPLC-MS (Method C): m/z: 351 (M+1); Rt=3.74 min.

Example 557 General Procedure (J)9-(3-Cyanobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

HPLC-MS (Method C): m/z: 351 (M+1); Rt=3.73 min.

Example 558 General Procedure (J)9-(5-Chloro-2-methoxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

¹H-NMR (DMSO-d₆): δ 8.87 (1H, d), 8.35 (1H, d), 8.10 (1H, dd), 7.73 (1H,d), 7.59 (1H, d), 7.49 (1H, t), 7.29 (1H, t), 7.27 (1H, dd), 7.11 (1H,d), 6.51 (1H, d), 5.63 (2H, s), 3.88 (3H, s); HPLC-MS (Method C): m/z:390 (M+1); Rt=4.37 min.

Example 559 General Procedure (J)N-Phenyl-2-[3-(2H-tetrazol-5-yl)carbazol-9-yl]acetamide

¹H-NMR (DMSO-d₆): δ 10.54 (1H, s), 8.87 (1H, bs), 8.27 (1H, d), 8.12(1H, bd), 7.83 (1H, d), 7.66 (1H, d), 7.61 (2H, d), 7.53 (1H, t), 7.32(1H, t), 7.32 (2H, t), 7.07 (1H, t), 5.36 (2H, s); HPLC-MS (Method C):m/z: 369 (M+1); Rt=3.44 min.

Example 560 General Procedure (J)N-Butyl-2-[3-(2H-tetrazol-5-yl)carbazol-9-yl]acetamide

¹H-NMR (DMSO-d₆): δ 8.85 (1H, d), 8.31 (1H, t), 8.25 (1H, d), 8.10 (1H,dd), 7.75 (1H, d), 7.58 (1H, d), 7.52 (1H, t), 7.30 (1H, t), 5.09 (2H,s), 3.11 (2H, q), 1.42 (2H, quint), 1.30 (2H, sext), 0.87 (3H, t);HPLC-MS (Method C): m/z: 349 (M+1); Rt=3.20 min.

Example 561 General Procedure (J)9-(2,4-Dichlorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

¹H-NMR (DMSO-d₆): δ 8.92 (1H, d), 8.32 (1H, d), 8.09 (1H, dd), 7.76 (1H,d), 7.74 (1H, d), 7.58 (1H, d), 7.51 (1H, t), 7.33 (1H, t), 7.23 (1H,dd), 6.42 (1H, d), 5.80 (2H, s); HPLC-MS (Method B): m/z: 394 (M+1);Rt=5.87 min.

Example 562 General Procedure (J)9-(2-Methylbenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

¹H-NMR (DMSO-d₆): δ 8.92 (1H, d), 8.32 (1H, d), 8.08 (1H, dd), 7.72 (1H,d), 7.55 (1H, d), 7.48 (1H, t), 7.32 (1H, t), 7.26 (1H, d), 7.12 (1H,t), 6.92 (1H, t), 6.17 (1H, d), 5.73 (2H, s), 2.46 (3H, s); HPLC-MS(Method B): m/z: 340 (M+1); Rt=5.30 min.

Example 563 General Procedure (J)9-(3-Nitrobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

HPLC-MS (Method C): m/z: 371 (M+1); Rt=3.78 min.

Example 564 General Procedure (J)9-(3,4-Dichlorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

HPLC-MS (Method B): m/z: 394 (M+1); Rt=5.62 min.

Example 565 General Procedure (J)9-(2,4-Difluorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

¹H-NMR (DMSO-d₆): δ 8.89 (1H, d), 8.29 (1H, d), 8.11 (1H, dd), 7.88 (1H,d), 7.69 (1H, d), 7.52 (1H, t), 7.36-7.24 (2H, m), 7.06-6.91 (2H, m),5.78 (2H, s); HPLC-MS (Method B): m/z: 362 (M+1); Rt=5.17 min.

Example 566 General Procedure (J)9-(3,5-Difluorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

¹H-NMR (DMSO-d₆): δ 8.90 (1H, bs), 8.31 (1H, d), 8.13 (1H, bd), 7.90(1H, d), 7.73 (1H, d), 7.54 (1H, t), 7.34 (1H, t), 7.14 (1H, t), 6.87(2H, bd), 5.80 (2H, s); HPLC-MS (Method B): m/z: 362 (M+1); Rt=5.17 min.

Example 567 General Procedure (J)9-(3,4-Difluorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

¹H-NMR (DMSO-d₆): δ 8.89 (1H, bs), 8.29 (1H, d), 8.12 (1H, bd), 7.92(1H, d), 7.74 (1H, d), 7.54 (1H, t), 7.42-7.25 (3H, m), 6.97 (1H, bm),5.75 (2H, s); HPLC-MS (Method B): m/z: 362 (M+1); Rt=5.17 min.

Example 568 General Procedure (J)9-(3-Iodobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

HPLC-MS (Method B): m/z: 452 (M+1); Rt=5.50 min.

Example 569 General Procedure (J)3-(2H-Tetrazol-5-yl)-9-[3-(trifluoromethyl)benzyl]-9H-carbazole

¹H-NMR (DMSO-d₆): δ 8.89 (1H, d), 8.30 (1H, d), 8.11 (1H, dd), 7.90 (1H,d), 7.72 (1H, d), 7.67 (1H, bs), 7.62 (1H, bd), 7.53 (1H, t), 7.50 (1H,bt), 7.33 (1H, bd), 7.32 (1H, t), 5.87 (2H, s); HPLC-MS (Method B): m/z:394 (M+1); Rt=5.40 min.

Example 570 General Procedure (J)N-(4-Carboxyphenyl)-2-[3-(2H-tetrazol-5-yl)carbazol-9-yl]acetamide

3.6 fold excess sodium hydride was used.

HPLC-MS (Method B): m/z: 413 (M+1); Rt=3.92 min.

Example 571 General Procedure (J)N-(2-Propyl)-2-[3-(2H-tetrazol-5-yl)carbazol-9-yl]acetamide

HPLC-MS (Method B): m/z: 335 (M+1); Rt=3.70 min.

Example 572 General Procedure (J)N-Benzyl-N-phenyl-2-[3-(2H-tetrazol-5-yl)carbazol-9-yl]acetamide

HPLC-MS (Method B): m/z: 459 (M+1); Rt=5.37 min.

Example 573 General Procedure (J)N-[4-(2-Methyl-2-propyl)phenyl]-2-[3-(2H-tetrazol-5-yl)carbazol-9-yl]acetamide

HPLC-MS (Method B): m/z: 425 (M+1); Rt=5.35 min.

Example 574 General Procedure (J)N-Phenethyl-2-[3-(2H-tetrazol-5-yl)carbazol-9-yl]acetamide

HPLC-MS (Method C): m/z: 397 (M+1); Rt=3.43 min.

Example 575 General Procedure (J)3-(2H-Tetrazol-5-yl)-9-[2-(trifluoromethyl)benzyl]-9H-carbazole

HPLC-MS (Method C): m/z: 394 (M+1); Rt=4.44 min.

Example 576 General Procedure (J)9-[2-Fluoro-6-(trifluoromethyl)benzyl]-3-(2H-tetrazol-5-yl)-9H-carbazole

HPLC-MS (Method C): m/z: 412 (M+1); Rt=4.21 min.

Example 577 General Procedure (J)9-[2,4-Bis(trifluoromethyl)benzyl)]-3-(2H-tetrazol-5-yl)-9H-carbazole

HPLC-MS (Method C): m/z: 462 (M+1); Rt=4.82 min.

Example 578 General Procedure (J)3-(2H-Tetrazol-5-yl)-9-(2,4,6-trimethylbenzyl)-9H-carbazole

HPLC-MS (Method C): m/z: 368 (M+1); Rt=4.59 min.

Example 579 General Procedure (J)9-(2,3,5,6-Tetramethylbenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

HPLC-MS (Method C): m/z: 382 (M+1); Rt=4.47 min.

Example 580 General Procedure (J)9-[(Naphthalen-1-yl)methyl]-3-(2H-tetrazol-5-yl)-9H-carbazole

HPLC-MS (Method C): m/z: 376 (M+1); Rt=4.43 min.

Further preferred compounds of the invention that may be preparedaccording to general procedure (J) includes:

The following preferred compounds of the invention may be prepared eg.from 9-(4-bromobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole (example 542)or from 9-(3-bromobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole (example536) and aryl boronic acids via the Suzuki coupling reaction eg asdescribed in Littke, Dai & Fu J. Am. Chem. Soc., 2000, 122, 4020-8 (orreferences cited therein), or using the methodology described in generalprocedure (E), optionally changing the palladium catalyst tobis(tri-tert-butylphosphine)palladium (0).

General Procedure (K) for Preparation of Compounds of General FormulaI₁₀:

wherein AR2 is as defined above.

The general procedure (K) is further illustrated by the followingexample:

Example 581 General Procedure (K)1-Benzyl-5-(2H-tetrazol-5-yl)-1H-indole

5-Cyanoindole (1.0 g, 7.0 mmol) was dissolved in N,N-dimethylformamide(14 mL) and cooled in an ice-water bath. Sodium hydride (0.31 g, 60%,7.8 mmol) was added, and the resulting suspension was stirred for 30min. Benzyl chloride (0.85 mL, 0.94 g, 7.4 mmol) was added, and thecooling was discontinued. The stirring was continued for 65 hours atroom temperature. Water (150 mL) was added, and the mixture wasextracted with ethyl acetate (3×25 mL). The combined organic phases werewashed with brine (30 mL) and dried with sodium sulfate (1 hour).Filtration and concentration yielded the crude material. Purification byflash chromatography on silica gel eluting with ethylacetate/heptanes=1:3 afforded 1.60 g 1-benzyl-1H-indole-5-carbonitrile.

HPLC-MS (Method C): m/z: 233 (M+1); Rt=4.17 min.

1-Benzyl-1H-indole-5-carbonitrile was transformed into1-benzyl-5-(2H-tetrazol-5-yl)-1H-indole by the method described ingeneral procedure (J) and in example 401. Purification was done by flashchromatography on silica gel eluting with dichloromethane/methanol=9:1.

HPLC-MS (Method C): m/z: 276 (M+1); Rt=3.35 min.

The compounds in the following examples were prepared by the sameprocedure.

Example 582 General Procedure (K)1-(4-Bromobenzyl)-5-(2H-tetrazol-5-yl)-1H-indole

HPLC-MS (Method C): m/z: 354 (M+1); Rt=3.80 min.

Example 583 General Procedure (K)1-(4-Phenylbenzyl)-5-(2H-tetrazol-5-yl)-1H-indole

¹H-NMR (200 MHz, DMSO-d₆): δ=5.52 (2H, s), 6.70 (1H, d), 7.3-7.45 (6H,m), 7.6 (4H, m), 7.7-7.8 (2H, m), 7.85 (1H, dd), 8.35 (1H, d).

Calculated for C₂₂H₁₇N₅, H₂O: 73.32% C; 5.03% H; 19.43% N. Found: 73.81%C; 4.90% H; 19.31% N.

Example 584 General Procedure (K) 5-(2H-Tetrazol-5-yl)-1H-indole

5-(2H-Tetrazol-5-yl)-1H-indole was prepared from 5-cyanoindole accordingto the method described in example 401.

HPLC-MS (Method C): m/z: 186 (M+1); Rt=1.68 min.

Example 585 General Procedure (K)1-Benzyl-4-(2H-tetrazol-5-yl)-1H-indole

1-Benzyl-1H-indole-4-carbonitrile was prepared from 4-cyanoindoleaccording to the method described in example 581.

HPLC-MS (Method C): m/z: 233 (M+1); Rt=4.24 min.

1-Benzyl-4-(2H-tetrazol-5-yl)-1H-indole was prepared from1-benzyl-1H-indole-4-carbonitrile according to the method described inexample 401.

HPLC-MS (Method C): m/z: 276 (M+1); Rt=3.44 min.

Further preferred compounds of the invention that may be preparedaccording to general procedure (K) includes:

The following preferred compounds of the invention may be prepared eg.from 1-(4-bromobenzyl)-5-(2H-tetrazol-5-yl)-1H-indole (example 532) orfrom the analogue 1-(3-bromobenzyl)-5-(2H-tetrazol-5-yl)-1H-indole andaryl boronic acids via the Suzuki coupling reaction eg as described inLittke, Dai & Fu J. Am. Chem. Soc., 2000, 122, 4020-8 (or referencescited therein), or using the methodology described in general procedure(E), optionally changing the palladium catalyst tobis(tri-tert-butylphosphine)palladium (0).

General Procedure (L) for Preparation of Compounds of General FormulaI₁₁:

The general procedure (L) is further illustrated by the followingexample:

Example 586 General Procedure (L)1-Benzoyl-5-(2H-tetrazol-5-yl)-1H-indole

To a solution of 5-cyanoindole (1.0 g, 7.0 mmol) in dichloromethane (8mL) was added 4-(dimethylamino)pyridine (0.171 g, 1.4 mmol),triethylamine (1.96 mL, 1.42 g, 14 mmol) and benzoyl chloride (0.89 mL,1.08 g, 7.7 mmol). The resulting mixture was stirred for 18 hours atroom temperature. The mixture was diluted with dichloromethane (80 mL)and washed consecutively with a saturated solution of sodiumhydrogencarbonate (40 mL) and brine (40 mL). The organic phase was driedwith magnesium sulfate (1 hour). Filtration and concentration furnishedthe crude material which was purified by flash chromatography on silicagel, eluting with ethyl acetate/heptanes=2:3.1-Benzoyl-1H-indole-5-carbonitrile was obtained as a solid.

HPLC-MS (Method C): m/z: 247 (M+1); Rt=4.07 min.

1-Benzoyl-1H-indole-5-carbonitrile was transformed into1-benzoyl-5-(2H-tetrazol-5-yl)-1H-indole by the method described inexample 401.

HPLC (Method C): Rt=1.68 min.

The compound in the following example was prepared by the sameprocedure.

Example 587 General Procedure (L)1-Benzoyl-4-(2H-tetrazol-5-yl)-1H-indole

1-Benzoyl-1H-indole-4-carbonitrile was prepared from 4-cyanoindoleaccording to the method described in example 586.

HPLC-MS (Method C): m/z: 247 (M+1); Rt=4.24 min.

1-Benzoyl-4-(2H-tetrazol-5-yl)-1H-indole was prepared from1-benzoyl-1H-indole-4-carbonitrile according to the method described inexample 401.

HPLC (Method C); Rt=1.56 min.

The following known and commercially available compounds do all bind tothe His B10 Zn²⁺ site of the insulin hexamer:

Example 588 1-(4-Fluorophenyl)-5-(2H-tetrazol-5-yl)-1H-indole

Example 589 1-Amino-3-(2H-tetrazol-5-yl)benzene

Example 590 1-Amino-4-(2H-tetrazol-5-yl)benzene

A mixture of 4-aminobenzonitrile (10 g, 84.6 mmol), sodium azide (16.5g, 254 mmol) and ammonium chloride (13.6 g, 254 mmol) in DMF was heatedat 125° C. for 16 hours. The cooled mixture was filtered and thefiltrate was concentrated in vacuo. The residue was added water (200 mL)and diethyl ether (200 mL) which resulted in crystallisation. Themixture was filtered and the solid was dried in vacuo at 40° C. for 16hours to afford 5-(4-aminophenyl)-2H-tetrazole.

¹H NMR DMSO-d₆): δ=5.7 (3H, bs), 6.69 (2H, d), 7.69 (2H, d). HPLC-MS(Method C): m/z: 162 (M+1); Rt=0.55 min.

Example 591 1-Nitro-4-(2H-tetrazol-5-yl)benzene

Example 592 1-Bromo-4-(2H-tetrazol-5-yl)benzene

General Procedure (M) for Solution Phase Preparation of Amides ofGeneral Formula I₁₂:

wherein A, B¹, B² are as defined above, R is hydrogen, optionallysubstituted aryl or C₁₋₈-alkyl and R′ is hydrogen or C₁₋₄-alkyl.

A-B¹—B²—CO₂H may be prepared eg by general procedure (D) or by othersimilar procedures described herein, or may be commercially available.

The procedure is further illustrated in the following example 593:

Example 593 General Procedure (M)N-(4-Chlorobenzyl)-2-[3-(2,4-dioxothiazolidin-5-ylidenemethyl)-1H-indol-1-yl]acetamide

[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indol-1-yl]acetic acid (example300, 90.7 mg, 0.3 mmol) was dissolved in NMP (1 mL) and added to amixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydrochloride(86.4 mg, 0.45 mmol) and 1-hydroxybenzotriazol (68.8 mg, 0.45 mmol) inNMP (1 mL). The resulting mixture was shaken at RT for 2 h.4-Chlorobenzylamine (51 mg, 0.36 mmol) and DIPEA (46.4 mg, 0.36 mmol) inNMP (1 mL) were added to the mixture resulting mixture shaken at RT for2 days. Subsequently ethyl acetate (10 mL) was added and the resultingmixture washed with 2×10 mL water followed by saturated ammoniumchloride (5 mL). The organic phase was evaporated to dryness giving 75mg (57%) of the title compound.

HPLC-MS (Method C): m/z: 426 (M+1); Rt.=3.79 min.

Example 594 General Procedure (M) 1H-Benzotriazole-5-carboxylic acid4-chlorobenzylamide

HPLC-MS (Method B): m/z: 287 (M+1); Rt=4.40 min.

Example 595 General Procedure (M)N-(4-Chlorobenzyl)-4-[2-chloro-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyramide

HPLC-MS (Method A): m/z: 465 (M+1); Rt=4.35 min.

Example 596 General Procedure (M)N-(4-Chlorobenzyl)-4-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyramide

HPLC-MS (Method A): m/z: 431 (M+1); Rt=3.68 min.

Example 597 General Procedure (M)2-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]-N-(4-chlorobenzyl)acetamide

HPLC-MS (Method A): m/z: 483 (M+1); Rt=4.06 min.

Example 598 General Procedure (M)N-(4-Chlorobenzyl)-2-[3-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]acetamide

HPLC-MS (Method A): m/z: 403 (M+1); Rt=4.03 min.

Example 599 General Procedure (M)N-(4-Chlorobenzyl)-3-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenyl]acrylamide

HPLC-MS (Method A): m/z: 399 (M+1); Rt=3.82.

Example 600 General Procedure (M)N-(4-Chlorobenzyl)-4-[3-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyramide

HPLC-MS (Method A): m/z: 431 (M+1); Rt=3.84 min.

Example 601 General Procedure (M)4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]-N-(4-chlorobenzyl)butyramide

HPLC-MS (Method A): m/z: 511 (M+1)); Rt=4.05 min.

Example 602 General Procedure (M)4-[2-Bromo-4-(4-oxo-2-thioxothiazolidin-5-ylidenemethyl)-phenoxy]-N-(4-chlorobenzyl)-butyramide

HPLC-MS (Method A): m/z: 527 (M+1); Rt=4.77 min.

Example 603 General Procedure (M)N-(4-Chlorobenzyl)-2-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]acetamide

HPLC-MS (Method C): m/z: 431 (M+1); Rt.=4.03 min.

Example 604 General Procedure (M)N-(4-Chlorobenzyl)-3-[3-(2,4-dioxothiazolidin-5-ylidenemethyl)-1H-indol-1-yl]propionamide

HPLC-MS (Method C): m/z: 440 (M+1); Rt.=3.57 min.

Example 605 General Procedure (M)N-(4-Chlorobenzyl)-4-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyramide

HPLC-MS (Method C): m/z: 481 (M+1); Rt=4.08 min.

Example 606 General Procedure (M)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)-naphthalen-1-yloxy]-N-hexylbutyramide

HPLC-MS (Method C): m/z: 441 (M+1); Rt=4.31 min.

Example 607 General Procedure (M)N-(4-Chlorobenzyl)-4-[3-(2H-tetrazol-5-yl)carbazol-9-ylmethyl]benzamide

HPLC-MS (Method C): m/z: 493 (M+1); Rt=4.19 min.

Example 608 General Procedure (M)N-(4-Chlorobenzyl)-3-[3-(2H-tetrazol-5-yl)carbazol-9-ylmethyl]benzamide

HPLC-MS (Method C): m/z: 493 (M+1); Rt=4.20 min.

Example 6094-({[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indole-7-carbonyl]amino}methyl)benzoicacid Methyl Ester

HPLC-MS (Method C): m/z: 436 (M+1); Rt.=3.55 min.

The commercially available compounds in the following examples do allbind to the HisB10 Zn²⁺ site:

Example 610 1-(4-Bromo-3-methylphenyl)-1,4-dihydrotetrazole-5-thione

Example 611 1-(4-Iodophenyl)-1,4-dihydrotetrazole-5-thione

Example 612 1-(2,4,5-Trichlorophenyl)-1H-tetrazole-5-thiol

Example 613 1-(2,6-Dimethylphenyl)-1,4-dihydrotetrazole-5-thione

Example 614 1-(2,4,6-Trimethylphenyl)-1,4-dihydrotetrazole-5-thione

Example 615 1-(4-Dimethylaminophenyl)-1H-tetrazole-5-thiol

Example 616 1-(3,4-Dichlorophenyl)-1,4-dihydro-1H-tetrazole-5-thione

Example 617 1-(4-Propylphenyl)-1,4-dihydro-1H-tetrazole-5-thione

Example 618 1-(3-Chlorophenyl)-1,4-dihydro-1H-tetrazole-5-thione

Example 619 1-(2-Fluorophenyl)-1,4-dihydro-1H-tetrazole-5-thione

Example 620 1-(2,4-Dichlorophenyl)-1,4-dihydro-1H-tetrazole-5-thione

Example 6211-(4-Trifluoromethoxyphenyl)-1,4-dihydro-1H-tetrazole-5-thione

Example 622 N-[4-(5-Mercaptotetrazol-1-yl)-phenyl]-acetamide

Example 623 1-(4-Chlorophenyl)-1,4-dihydrotetrazole-5-thione

Example 624 1-(4-Methoxyphenyl)-1,4-dihydrotetrazole-5-thione

Example 6251-(3-Fluoro-4-pyrrolidin-1-ylphenyl)-1,4-dihydrotetrazole-5-thione

Preparation of 1-aryl-1,4-dihydrotetrazole-5-thiones (or the tautomeric1-aryltetrazole-5-thiols) is described in the literature (eg. by Kauer &Sheppard, J. Org. Chem., 32, 3580-92 (1967)) and is generally performedeg. by reaction of aryl-isothiocyanates with sodium azide followed byacidification

1-Aryl-1,4-dihydrotetrazole-5-thiones with a carboxylic acid tethered tothe aryl group may be prepared as shown in the following scheme:

Step 1 is a phenol alkylation and is very similar to steps 1 and 2 ofgeneral procedure (D) and may also be prepared similarly as described inexample 303.

Step 2 is a reduction of the nitro group. SnCl₂, H₂ over Pd/C and manyother procedures known to those skilled in the art may be utilised.

Step 3 is formation of an arylisothiocyanate from the correspondinganiline. As reagents CS₂, CSCl₂, or other reagents known to thoseskilled in the art, may be utilised.

Step 4 is a conversion to mercaptotetrazole as described above.

Preferred compounds of the invention includes:

Example 626 4-(4-Hydroxyphenyl)-1H-[1,2,3]triazole-5-carbonitrile

Phenylsulphonyl acetonitrile (2.0 g, 11.04 mmol) was mixed with4-hydroxybenzaldehyde (1.35 g, 11.04 mmol) in DMF (10 mL) and toluene(20 mL). The mixture was refluxed for 3 hours and subsequentlyevaporated to dryness in vacuo. The residue was treated with diethylether and toluene. The solid formed was filtered to afford 2.08 g (66%)of 2-benzenesulfonyl-3-(4-hydroxyphenyl)acrylonitrile.

HPLC-MS (Method C): m/z: 286 (M+1); Rt.=3.56 min.

A mixture of 2-benzenesulfonyl-3-(4-hydroxyphenyl)acrylonitrile (2.08 g,7.3 mmol) and sodium azide (0.47 g, 7.3 mmol) in DMF (50 mL) was heatedat reflux temperature 2 hours. After cooling, the mixture was poured onice. The mixture was evaporated in vacuo to almost dryness and toluenewas added. After filtration, the organic phase was evaporated in vacuo.The residue was purified by silicagel chromatography eluting with amixture of ethyl acetate and heptane (1:2). This afforded 1.2 g (76%) ofthe title compound.

¹H NMR (DMSO-d₆): 10.2 (broad, 1H); 7.74 (d, 2H); 6.99 (d, 2H); 3.6-3.2(broad, 1H). HPLC-MS (Method C) m/z:=187 (M+1); Rt.=1.93 min

The compounds in the following examples are commercially available andmay be prepared using a similar methodology:

Example 6274-(4-Trifluoromethoxyphenyl)-1H-[1,2,3]triazole-5-carbonitrile

Example 628 4-Benzo[1,3]dioxol-5-yl-1H-[1,2,3]triazole-5-carbonitrile

Example 6294-(3-Trifluoromethylphenyl)-1H-[1,2,3]triazole-5-carbonitrile

Example 630 4-Pyridin-3-yl-1H-[1,2,3]triazole-5-carbonitrile

Example 631 4-(2,6-Dichlorophenyl)-1H-[1,2,3]triazole-5-carbonitrile

Example 632 4-Thiophen-2-yl-1H-[1,2,3]triazole-5-carbonitrile

Example 633 3,5-Dimethylisoxazole-4-carboxylic acid4-(5-cyano-1H-[1,2,3]triazol-4-yl)phenyl Ester

Example 634 3,3-Dimethyl-butyric acid4-(5-cyano-1H-[1,2,3]triazol-4-yl)phenyl Ester

Example 635 4-Methyl-[1,2,3]thiadiazole-5-carboxylic acid4-(5-cyano-1H-[1,2,3]triazol-4-yl)phenyl Ester

Example 636 4-Chlorobenzoic acid4-(5-cyano-1H-[1,2,3]triazol-4-yl)phenyl Ester

Example 637 4-(3-Phenoxyphenyl)-1H-[1,2,3]triazole-5-carbonitrile

Example 6384-(5-Bromo-2-methoxyphenyl)-1H-[1,2,3]triazole-5-carbonitrile

Example 6394-(2-Chloro-6-fluorophenyl)-1H-[1,2,3]triazole-5-carbonitrile

The following cyanotriazoles are also preferred compounds of theinvention:

-   4-(2-Chloro-6-fluorophenyl)-1H-[1,2,3]triazole-5-carbonitrile.-   Terephthalic acid mono[4-(5-cyano-1H-[1,2,3]triazol-4-yl)phenyl]    ester.-   N-[4-(5-cyano-1H-[1,2,3]triazol-4-yl)-phenyl]terephthalamic acid-   4-(4-Octyloxyphenyl)-1H-[1,2,3]triazole-5-carbonitrile-   4-(Styrylphenyl))-1H-[1,2,3]triazole-5-carbonitrile.-   4-(4′-Trifluoromethylbiphenyl-4-yl)-1H-[1,2,3]triazole-5-carbonitrile.-   4-(4′-Chlorobiphenyl-4-yl)-1H-[1,2,3]triazole-5-carbonitrile.-   4-(4′-Methoxybiphenyl-4-yl)-1H-[1,2,3]triazole-5-carbonitrile.-   4-(1-Naphthyl)-1H-[1,2,3]triazole-5-carbonitrile.-   4-(9-Anthranyl)-1H-[1,2,3]triazole-5-carbonitrile.-   4-(4-Methoxy-1-naphthyl)-1H-[1,2,3]triazole-5-carbonitrile.-   4-(4-Aminophenyl)-1H-[1,2,3]triazole-5-carbonitrile.-   4-(2-Naphthyl)-1H-[1,2,3]triazole-5-carbonitrile.    General Procedure (N) for Preparation of Compounds of General    Formula I₁₃:

wherein

-   -   n is 1 or 3-20,    -   AR¹ is as defined above,    -   R″ is a standard carboxylic acid protecting group, such as        C₁-C₆-alkyl or benzyl and Lea is a leaving group, such as        chloro, bromo, iodo, methanesulfonyloxy, toluenesulfonyloxy or        the like.

This procedure is very similar to general procedure (D), steps 1 and 2are identical.

Steps 3 and 4 are described in the literature (eg Beck & Gûnther, Chem.Ber., 106, 2758-66 (1973))

Step 3 is a Knoevenagel condensation of the aldehyde obtained in step 2with phenylsulfonylacetonitrile and step 4 is a reaction of thevinylsulfonyl compound obtained in step 3 with sodium azide. Thisreaction is usually performed in DMF at 90-110° C.

The following compounds may be prepared according to this generalprocedure (N):

-   4-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)butyric acid:

-   2-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)acetic acid:

-   4-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)butyric acid ethyl    ester-   5-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)pentanoic acid-   8-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)octanoic acid-   10-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)decanoic acid-   12-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)dodecanoic acid    General Procedure (O) for Preparation of Compounds of General    Formula I₇:

(SEQ ID NOS: 20-23 and 23, respectively, in order or appearance)

wherein PS is polymeric support, a Tentagel S RAM resin, n is 1-20, m is0-5, and p is 0 or 1.

The compounds of the invention of general formula (I₂) can be preparedby means of standard peptide chemistry (General procedure H), e.g. in0.5 mmol scale, using Fmoc strategy and HOAt or HOBT activated aminoacids. The compounds prepared in the following examples according toGeneral procedure (O) were all isolated as the TFA salts. This procedureis further illustrated in the following:

Typically, 2 gram of Fmoc Tentagel S RAM resin (Rapp Polymere, Tubingen)with substitution 0.25 mmol/g was washed with NMP then treated with 25%piperidine in NMP for 30 min followed by wash with NMP which renders theresin ready for coupling.

Step wise coupling of Fmoc-Arginine (Fmoc-Arg(Pmc)-OH), Fmoc-Glycine(Fmoc-Gly-OH) and Fmoc-4-aminobenzoic acid (Fmoc-4-Abz-OH):

To 2 mmol of Fmoc-L-Arg(Pmc)-OH (Novabiochem) was added 3.33 ml 0.6MHOAt in NMP (Perseptives) or 0.6M HOBT in NMP (Novabiochem) containing0.2% bromphenolblue as indicator and added 330 μl ofdiisopropylcarbodiimide DIC (Fluka) and the solution was then added tothe resin. After coupling for minimum 1 hour, or when the blue colourdisappeared, the resin was washed with NMP and the Fmoc group wasdeprotected with 25% piperidine in NMP for 20 minutes followed by washwith NMP. This stepwise assembling of the arginine residues was repeatedto give 3, 4, 5 or 6 arginines on the resin. The Fmoc-Glycine(Novabiochem) and Fmoc-4-aminobenzoic acid (Fluka and Neosystems) werecoupled using the same procedure as described for Fmoc-Arg(Pmc)-OH.

Coupling of A-OH, e.g. 1H-benzotriazole-5-carboxylic acid on Gly.

When A-OH, e.g. 1H-benzotriazole-5-carboxylic acid (Aldrich) was coupledon a glycine or arginine residue the coupling procedure was as describedabove.

Coupling of A-OH, e.g. 1H-benzotriazole-5-carboxylic acid on Abz or4-Apac:

Due to the lower nucleophilicity of the amino group in Abz the followingprocedure was necessary. To 4 mmol of A-OH, e.g.1H-benzotriazole-5-carboxylic acid was added 6.66 ml of a solution of0.6M HOAt, 0.2 mmol dimethylaminopyridine (DMAP) and 4-mmol DIC and wasthen added to the resin and allowed to react overnight.

Introduction of Fragment 4-Apac Instead of 4-Abz:

4-Nitrophenoxyacetic acid may be coupled on a glycine or arginineresidue using DIC and HOBT/HOAt as described above. Subsequent reductionof the nitro group may be done using SnCl₂ in NMP or DMF e.g. asdescribed by Tumelty et al. (Tet. Lett., (1998) 7467-70).

Cleavage of the Peptides from the Resin.

After synthesis the resin was washed extensively with diethyl ether anddried. To 1 gram of the peptidyl resin was added 25 ml of a TFA solutioncontaining 5% thioanisole, 5% ethanol, 5% phenol and 2%triisopropylsilane and allowed to react for 2 hours. The TFA solutionwas filtered and concentrated with argon flow for approximately 30minutes. Then diethylether ca. 5-7 times the residual volume of TFA wasadded and the peptide precipitate was extracted in 10% AcOH and washed 5times with diethyl ether and lyophilized.

RP-HPLC analysis and purification: The crude products were analysed onRP-HPLC C18 column (4.6×250 mm) using one of two gradients (see table 1and 2), temperature 25° C., wavelength 214 nm and flow rate 1 ml/minwith A-buffer 0.15% (^(w)/_(w)) TFA in H₂O and B-Buffer (87.5%(^(w)/_(w)) MeCN, 0.13% (^(w)/_(w)) TFA in H₂O)

The products were purified on preparative RP-HPLC C18 column (2×25 cm)using a gradient (variable, see e.g examples 640 to 643643643),temperature 25° C., wavelength 214 nm and flow rate 6 ml/min withA-buffer 0.15% (^(w)/_(w)) TFA in H₂O and B-Buffer (87.5% (^(w)/_(w))MeCN, 0.13% (^(w)/_(w)) TFA in H₂O) and verified by mass spectrometry(MALDI).

TABLE 1 Time (min.) Flow (ml/min) ( % A % B 0 1.00 95.0 5.0 30.00 1.0080.0 20.0 35.00 1.00 0.0 100.0 40.00 1.00 0.0 100.0 45.00 1.00 95.0 5.0

TABLE 2 Time (min.) Flow (ml/min) % A % B 0 1.00 95.0 5.0 30.00 1.0040.0 60.0 31.00 1.00 0.00 100.0 35.00 1.00 0.00 100.0 36.00 1.00 95.05.0

The following examples were prepared using this general procedure (O).

Example 640 General Procedure (O)Benzotriazol-5-ylcarbonyl-Gly₂-Arg₃-NH₂ (BT-G₂R₃) (SEQ ID NO: 6)

MS (MALDI): m/z: 746.7 g/mol; calculated: 744.2 g/mol.

HPLC gradient:

Time (min) Flow (ml/min) % A % B 0.00 6.00 90.0 10.0 120.00 6.00 90.010.0 121.00 0.10 90.0 10.0

Example 641 General Procedure (O)Benzotriazol-5-ylcarbonyl-Gly₂-Arg₄-NH₂ (BT-G₂R₄) (SEQ ID NO: 2)

MS (MALDI): m/z: 903.0 g/mol; calculated: 900.6 g/mol.

HPLC gradient:

Time (min) Flow (ml/min) % A % B 0.00 6.00 95.0 5.0 30.00 6.00 80.0 20.035.00 6.00 0.0 100.0 40.00 6.00 0.0 100.0 45.00 6.00 95.0 5.0 64.00 6.0095.0 5.0

Example 642 General Procedure (O)Benzotriazol-5-ylcarbonyl-Gly₂-Arg₅-NH₂ (BT-G₂R₅) (SEQ ID NO: 1)

MS (MALDI): m/z: 1060.8 g/mol; calculated: 1057 g/mol.

HPLC gradient

Time (min) Flow (ml/min) % A % B 0.00 6.00 88.0 12.0 120.00 6.00 88.012.0 121.00 0.10 88.0 12.0

Example 643 General Procedure (O)Benzotriazol-5-ylcarbonyl-Gly₂-Arg₆-NH₂ (BT-G₂R₆) (SEQ ID NO: 5)

MS (MALDI): m/z: 1214.8 g/mol; calculated: 1213.4 g/mol.

HPLC gradient:

Time (min) Flow (ml/min) % A % B 0.00 6.00 88.0 12.0 120.00 6.00 88.012.0 121.00 0.10 88.0 12.0

Example 644 General Procedure (O)Benzotriazol-5-ylcarbonyl-4-Abz-Gly₂-Arg₅-NH₂ (BT-4-Abz-G₂R₅) (SEQ IDNO: 10)

MS (MALDI): m/z: 1176.7 g/mol; calculated: 1177.9 g/mol.

HPLC gradient:

Time (min) Flow (ml/min) % A % B 0.00 6.00 95.0 5.0 40.00 6.00 60.0 40.045.00 6.00 60.0 40.0 50.00 6.00 0.0 100.0 55.00 6.00 0.0 100.0 60.006.00 95.0 5.0

Example 645 General Procedure (O)Benzotriazol-5-ylcarbonyl-4-Abz-Gly-Arg₅-NH₂ (BT-4-Abz-GR₅) (SEQ ID NO:24)

MS (MALDI): m/z: 1122 g/mol; calculated: 1120.4 g/mol.

HPLC gradient:

Time (min) Flow (ml/min) % A % B 0.00 6.00 95.0 5.0 40.00 6.00 60.0 40.045.00 6.00 60.0 40.0 50.00 6.00 0.0 100.0 55.00 6.00 0.0 100.0 60.006.00 95.0 5.0

Example 646 General Procedure (O)Benzotriazol-5-ylcarbonyl-4-Abz-Arg₅-NH₂ (BT-4-Abz-R₅) (SEQ ID NO: 13)

MS (MALDI): m/z: 1064.3 g/mol; calculated: 1063.2 g/mol.

HPLC gradient:

Time (min) Flow (ml/min) % A % B 0.00 6.00 95.0 5.0 40.00 6.00 60.0 40.045.00 6.00 60.0 40.0 50.00 6.00 0.0 100.0 55.00 6.00 0.0 100.0 60.006.00 95.0 5.0General Procedure (P) for Preparation of Compounds of General FormulaI₈:

(SEQ ID NOS: 20-22, 22 and 22, respectively, in order or appearance)

wherein X, Y, R¹⁰, E, B¹, B² are as defined above,

p is 0 or 1,

m is 0-5 and

n is 1-20.

This general procedure is very similar to General procedure (O), wherebenzotriazole-5-carboxylic acid in the last step before cleavage fromthe resin is replaced with compounds optionally prepared according togeneral procedure (D):

Example 647 General Procedure (P)4-{2-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetylamino}benzoyl-Gly₂-Arg₅-NH₂(SEQ ID NO: 25)

Example 648 General Procedure (P)3-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenyl]acryloyl-Arg₅-NH₂ (SEQID NO: 26)

MS (MALDI): m/z: 1057.3 g/mol; calculated: 1055.3 g/mol.

Example 649 General Procedure (P)3-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenyl]acryloyl-Arg₄-NH₂ (SEQID NO: 27)

MS (MALDI): m/z: 899.1 g/mol; calculated: 901.6 g/mol.

Example 650 General Procedure (P)3-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenyl]acryloyl-Arg₃-NH₂

MS (MALDI): m/z: 746.2 g/mol; calculated: 742.9 g/mol.

Example 651 General Procedure (P)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg₅-NH₂ (SEQID NO: 26)

MS (MALDI): m/z: 1088.7 g/mol; calculated: 1087 g/mol.

Example 652 General Procedure (P)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg₄-NH₂ (SEQID NO: 27)

MS (MALDI): m/z: 933.0 g/mol; calculated: 931 g/mol.

Example 653 General Procedure (P)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg₃-NH₂

MS (MALDI): m/z: 776.9 g/mol; calculated: 774.0 g/mol.

Example 654 General Procedure (P)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethypnaphthalen-1-yloxy]butyryl-Arg₁₂-NH₂(SEQ ID NO: 28)

MS (MALDI): m/z: 2232.9.4 g/mol; calculated: 2230.3 g/mol.

Example 655 General Procedure (P)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg₈-NH₂(SEQ ID NO: 29)

MS (MALDI): m/z: 1607.4 g/mol; calculated: 1605.5 g/mol.

Example 656 General Procedure (P)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg₅-NH₂(SEQ ID NO: 26)

MS (MALDI): m/z: 1141.9 g/mol; calculated: 1137.4 g/mol.

Example 657 General Procedure (P)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyDnaphthalen-1-yloxy]butyryl-Arg₄-NH₂(SEQ ID NO: 27)

MS (MALDI): m/z: 985.4 g/mol; calculated: 981.2 g/mol.

Example 658 General Procedure (P)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg₃-NH₂

MS (MALDI): m/z: 828.5 g/mol; calculated: 825.0 g/mol.

The following compounds were prepared according to the methodologydescribed in general procedure (O) and (P):

Example 659 4-(2H-Tetrazol-5-yl)benzoyl-4-Abz-Gly₂-Arg₅-NH₂ (SEQ ID NO:19)

MS (MALDI): m/z: 1203.8 g/mol; calculated: 1203.8 g/mol.

Example 660 4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg₅-NH₂(SEQ ID NO: 26)

MS (MALDI): m/z: 1152.5 g/mol; calculated: 1149.3 g/mol.

Example 661 4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg₈-NH₂(SEQ ID NO: 29)

MS (MALDI): m/z: 1621.0 g/mol; calculated: 1617.5 g/mol.

Example 662 4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg₁₂-NH₂(SEQ ID NO: 28)

MS (MALDI): m/z: 2247.9 g/mol; calculated: 2242.3 g/mol.

Other preferred compounds of the invention that may be preparedaccording to general procedure (O) and/or general procedure (P)includes:

Building block from example 291:4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg₁₀-NH₂(SEQ ID NO: 30)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg₉-NH₂(SEQ ID NO: 31)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg₈-NH₂(SEQ ID NO: 29)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg₇-NH₂(SEQ ID NO: 32)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg₁₁-NH₂(SEQ ID NO: 33)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg₁₂-NH₂(SEQ ID NO: 28)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg₁₃-NH₂(SEQ ID NO: 34)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg₁₄-NH₂(SEQ ID NO: 35)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg₁₅-NH₂(SEQ ID NO: 36)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg₁₆-NH₂(SEQ ID NO: 37)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg₁₇-NH₂(SEQ ID NO: 38)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg₁₈-NH₂(SEQ ID NO: 39)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg₁₉-NH₂(SEQ ID NO: 40)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg₂₀-NH₂(SEQ ID NO: 41)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys₆-NH₂(SEQ ID NO: 42)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys₅-NH₂(SEQ ID NO: 43)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys₄-NH₂(SEQ ID NO: 44)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys₃-NH₂4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys₇-NH₂(SEQ ID NO: 45)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys₈-NH₂(SEQ ID NO: 46)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys₉-NH₂(SEQ ID NO: 47)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys₁₀-NH₂(SEQ ID NO: 48)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys₁₁-NH₂(SEQ ID NO: 49)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys₁₂-NH₂(SEQ ID NO: 50)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys₁₃-NH₂(SEQ ID NO: 51)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys₁₄-NH₂(SEQ ID NO: 52)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys₁₅-NH₂(SEQ ID NO: 53)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys₁₆-NH₂(SEQ ID NO: 54)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys₁₇-NH₂(SEQ ID NO: 55)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys₁₈-NH₂(SEQ ID NO: 56)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys₁₉-NH₂(SEQ ID NO: 57)4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys₂₀-NH₂(SEQ ID NO: 58) Building block from example 292:5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoyl-Arg₆-NH₂(SEQ ID NO: 59)5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoyl-Arg₅-NH₂(SEQ ID NO: 26)5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoyl-Arg₄-NH₂(SEQ ID NO: 27)5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoyl-Arg₃-NH₂Building block from page 164:6-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]hexanoyl-Arg₃-NH₂6-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]hexanoyl-Arg₄-NH₂(SEQ ID NO: 27)6-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]hexanoyl-Arg₅-NH₂(SEQ ID NO: 26) Building block from page 164:7-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]heptanoyl-Arg₃-NH₂7-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]heptanoyl-Arg₄-NH₂(SEQ ID NO: 27)7-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]heptanoyl-Arg₅-NH₂(SEQ ID NO: 26) Building block from page 164:8-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]octanoyl-Arg₃-NH₂8-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]octanoyl-Arg₄-NH₂(SEQ ID NO: 27)8-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]octanoyl-Arg₅-NH₂(SEQ ID NO: 26) Building block from page 164:10-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]decanoyl-Arg₃-NH₂10-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]decanoyl-Arg₄-NH₂(SEQ ID NO: 27)10-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]decanoyl-Arg₅-NH₂(SEQ ID NO: 26) Building block from page 164:11-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]undecanoyl-Arg₃-NH₂11-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]undecanoyl-Arg₄-NH₂(SEQ ID NO: 27)11-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]undecanoyl-Arg₅-NH₂(SEQ ID NO: 26) Building block from page 164:12-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]dodecanoyl-Arg₃-NH₂12-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]dodecanoyl-Arg₄-NH₂(SEQ ID NO: 27)12-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]dodecanoyl-Arg₅-NH₂(SEQ ID NO: 26) Building block from page 164:15-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentadecanoyl-Arg₃-NH₂15-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentadecanoyl-Arg₄-NH₂(SEQ ID NO: 27)15-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentadecanoyl-Arg₅-NH₂(SEQ ID NO: 26) Building block from example 298:2-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]acetyl-Arg₆-NH₂(SEQ ID NO: 59)2-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]acetyl-Arg₅-NH₂(SEQ ID NO: 26)2-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]acetyl-Arg₄-NH₂(SEQ ID NO: 27)2-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]acetyl-Arg₃-NH₂Building block from example 302:2-{5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzylidene]-4-oxo-2-thioxothiazolidin-3-yl}acetyl-Arg₆-NH₂ (SEQ ID NO: 59)2-{5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzylidene]-4-oxo-2-thioxothiazolidin-3-yl}acetyl-Arg₅-NH₂ (SEQ ID NO: 26)2-{5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzylidene]-4-oxo-2-thioxothiazolidin-3-yl}acetyl-Arg₄-NH₂ (SEQ ID NO: 27)2-{5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzylidene]-4-oxo-2-thioxothiazolidin-3-yl}acetyl-Arg₃-NH₂4-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]butyryl-Arg₆-NH₂(SEQ ID NO: 59)4-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]butyryl-Arg₅-NH₂(SEQ ID NO: 26)4-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]butyryl-Arg₄-NH₂(SEQ ID NO: 27)4-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]butyryl-Arg₃-NH₂15-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]pentadecanoyl-Arg₆-NH₂(SEQ ID NO: 59)15-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]pentadecanoyl-Arg₅-NH₂(SEQ ID NO: 26)15-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]pentadecanoyl-Arg₄-NH₂(SEQ ID NO: 27)15-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]pentadecanoyl-Arg₃-NH₂5-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoyl-Arg₆-NH₂(SEQ ID NO: 59)5-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoyl-Arg₅-NH₂(SEQ ID NO: 26)5-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoyl-Arg₄-NH₂(SEQ ID NO: 27)5-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoyl-Arg₃-NH₂Building block from example 284:3-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenyl]acryloyl-Arg₆-NH₂ (SEQID NO: 59) Building block from example 295:2-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg₆-NH₂(SEQ ID NO: 59)2-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg₅-NH₂(SEQ ID NO: 26)2-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg₄-NH₂(SEQ ID NO: 27)2-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg₃-NH₂8-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]octanoyl-Arg₆-NH₂(SEQ ID NO: 59)8-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]octanoyl-Arg₅-NH₂(SEQ ID NO: 26)8-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]octanoyl-Arg₄-NH₂(SEQ ID NO: 27)8-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]octanoyl-Arg₃-NH₂6-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]hexanoyl-Arg₆-NH₂(SEQ ID NO: 59)6-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]hexanoyl-Arg₅-NH₂(SEQ ID NO: 26)6-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]hexanoyl-Arg₄-NH₂(SEQ ID NO: 27)6-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]hexanoyl-Arg₃-NH₂Building block from example 288:4-[2-Chloro-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg₆-NH₂(SEQ ID NO: 59)4-[2-Chloro-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg₅-NH₂(SEQ ID NO: 26)4-[2-Chloro-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg₄-NH₂(SEQ ID NO: 27)4-[2-Chloro-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg₃-NH₂Building block from example 282:4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg₆-NH₂ (SEQID NO: 59) Building block from example 289:4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg₆-NH₂(SEQ ID NO: 59)4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg₅-NH₂(SEQ ID NO: 26)4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg₄-NH₂(SEQ ID NO: 27)4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg₃-NH₂11-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]undecanoyl-Arg₆-NH₂(SEQ ID NO: 59)11-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]undecanoyl-Arg₅-NH₂(SEQ ID NO: 26)11-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]undecanoyl-Arg₄-NH₂(SEQ ID NO: 27)11-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]undecanoyl-Arg₃-NH₂4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg₆-NH₂(SEQ ID NO: 59)4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg₅-NH₂(SEQ ID NO: 26)4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg₄-NH₂(SEQ ID NO: 27)4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg₃-NH₂Building block from example 286:4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzoyl-Arg₆-NH₂ (SEQ ID NO: 59)4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzoyl-Arg₅-NH₂ (SEQ ID NO: 26)4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzoyl-Arg₄-NH₂ (SEQ ID NO: 27)4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzoyl-Arg₃-NH₂ Building blockfrom example 285:2-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg₆-NH₂ (SEQID NO: 59)2-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg₅-NH₂ (SEQID NO: 26)2-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg₄-NH₂ (SEQID NO: 27)2-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg₃-NH₂Building block from example 283:2-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg₆-NH₂ (SEQID NO: 59)2-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg₅-NH₂ (SEQID NO: 26)2-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg₄-NH₂ (SEQID NO: 27)2-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg₃-NH₂Building block from example 296:4-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg₆-NH₂ (SEQID NO: 59)4-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg₅-NH₂ (SEQID NO: 26)4-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg₄-NH₂ (SEQID NO: 27)4-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg₃-NH₂Building block from example 290:4-[2-Bromo-4-(4-oxo-2-thioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg₆-NH₂(SEQ ID NO: 59)4-[2-Bromo-4-(4-oxo-2-thioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg₅-NH₂(SEQ ID NO: 26)4-[2-Bromo-4-(4-oxo-2-thioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg₄-NH₂(SEQ ID NO: 27)4-[2-Bromo-4-(4-oxo-2-thioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg₃-NH₂Building block from example 544:4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg₆-NH₂ (SEQ ID NO:59) 4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg₄-NH₂ (SEQ IDNO: 27) 4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg₃-NH₂4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg₇-NH₂ (SEQ ID NO:32) 4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg₈-NH₂ (SEQ IDNO: 29) 4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg₉-NH₂ (SEQID NO: 31) 4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg₁₀-NH₂(SEQ ID NO: 30)4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg₁₁-NH₂ (SEQ ID NO:33) 4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg₁₂-NH₂ (SEQ IDNO: 28) 4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg₁₃-NH₂(SEQ ID NO: 34)4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg₁₄-NH₂ (SEQ ID NO:35) 4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg₁₅-NH₂ (SEQ IDNO: 36) 4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg₁₆-NH₂(SEQ ID NO: 37)4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg₁₇-NH₂ (SEQ ID NO:38) 4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg₁₈-NH₂ (SEQ IDNO: 39) 4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg₁₉-NH₂(SEQ ID NO: 40)4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg₂₀-NH₂ (SEQ ID NO:41) Building block from page 251:4′-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]biphenyl-4-carbonyl-Arg₆-NH₂(SEQ ID NO: 59)4′-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]biphenyl-4-carbonyl-Arg₅-NH₂(SEQ ID NO: 26)4′-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]biphenyl-4-carbonyl-Arg₄-NH₂(SEQ ID NO: 27)4′-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]biphenyl-4-carbonyl-Arg₃-NH₂Building block from example 549:3-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg₆-NH₂ (SEQ ID NO:59) 3-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg₅-NH₂ (SEQ IDNO: 26) 3-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg₄-NH₂ (SEQID NO: 27) 3-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg₃-NH₂Building block from page 252:4′-[5-(2H-Tetrazol-5-yl)indol-1-ylmethyl]biphenyl-4-carbonyl-Arg₆-NH₂(SEQ ID NO: 59)4′-[5-(2H-Tetrazol-5-yl)indol-1-ylmethyl]biphenyl-4-carbonyl-Arg₅-NH₂(SEQ ID NO: 26)4′-[5-(2H-Tetrazol-5-yl)indol-1-ylmethyl]biphenyl-4-carbonyl-Arg₄-NH₂(SEQ ID NO: 27)4′-[5-(2H-Tetrazol-5-yl)indol-1-ylmethyl]biphenyl-4-carbonyl-Arg₃-NH₂Building block from example 412:4-(2H-Tetrazol-5-yl)benzoyl-Gly₂-Arg₆-NH₂ (SEQ ID NO: 59)4-(2H-Tetrazol-5-yl)benzoyl-Gly₂-Arg₅-NH₂ (SEQ ID NO: 26)4-(2H-Tetrazol-5-yl)benzoyl-Gly₂-Arg₄-NH₂ (SEQ ID NO: 27)4-(2H-Tetrazol-5-yl)benzoyl-Gly₂-Arg₃-NH₂ Building block from example355: [4-(7-Carboxy-6-hydroxynaphthalen-2-yl)phenyl]methyl-Arg₆-NH₂ (SEQID NO: 59) [4-(7-Carboxy-6-hydroxynaphthalen-2-yl)phenyl]methyl-Arg₅-NH₂(SEQ ID NO: 26)[4-(7-Carboxy-6-hydroxynaphthalen-2-yl)phenyl]methyl-Arg₄-NH₂ (SEQ IDNO: 27) [4-(7-Carboxy-6-hydroxynaphthalen-2-yl)phenyl]methyl-Arg₃-NH₂Building block from example 342:(7-Carboxy-6-hydroxynaphthalen-2-yl)methyl-Arg₆-NH₂ (SEQ ID NO: 59)(7-Carboxy-6-hydroxynaphthalen-2-yl)methyl-Arg₅-NH₂ (SEQ ID NO: 26)(7-Carboxy-6-hydroxynaphthalen-2-yl)methyl-Arg₄-NH₂ (SEQ ID NO: 27)(7-Carboxy-6-hydroxynaphthalen-2-yl)methyl-Arg₃-NH₂ Building block fromexample 342:4-[(7-Carboxy-6-hydroxynaphthalen-2-ylmethyl)amino]benzoyl-Arg₆-NH₂ (SEQID NO: 59)4-[(7-Carboxy-6-hydroxynaphthalen-2-ylmethyl)amino]benzoyl-Arg₅-NH₂ (SEQID NO: 26)4-[(7-Carboxy-6-hydroxynaphthalen-2-ylmethyl)amino]benzoyl-Arg₄-NH₂ (SEQID NO: 27)4-[(7-Carboxy-6-hydroxynaphthalen-2-ylmethyl)amino]benzoyl-Arg₃-NH₂4-[4-(5-Mercaptotetrazol-1-yl)benzoylamino]benzoyl-Arg₆-NH₂ (SEQ ID NO:59) 4-[4-(5-Mercaptotetrazol-1-yl)benzoylamino]benzoyl-Arg₅-NH₂ (SEQ IDNO: 26) 4-[4-(5-Mercaptotetrazol-1-yl)benzoylamino]benzoyl-Arg₄-NH₂ (SEQID NO: 27) 4-[4-(5-Mercaptotetrazol-1-yl)benzoylamino]benzoyl-Arg₃-NH₂4-[4-(5-Mercaptotetrazol-1-yl)phenoxy]butyryl-Arg₆-NH₂ (SEQ ID NO: 59)4-[4-(5-Mercaptotetrazol-1-yl)phenoxy]butyryl-Arg₅-NH₂ (SEQ ID NO: 26)4-[4-(5-Mercaptotetrazol-1-yl)phenoxy]butyryl-Arg₄-NH₂ (SEQ ID NO: 27)4-[4-(5-Mercaptotetrazol-1-yl)phenoxy]butyryl-Arg₃-NH₂4-[4-(5-Mercaptotetrazol-1-yl)naphthalen-1-yloxy]butyryl-Arg₆-NH₂ (SEQID NO: 59)4-[4-(5-Mercaptotetrazol-1-yl)naphthalen-1-yloxy]butyryl-Arg₅-NH₂ (SEQID NO: 26)4-[4-(5-Mercaptotetrazol-1-yl)naphthalen-1-yloxy]butyryl-Arg₄-NH₂ (SEQID NO: 27)4-[4-(5-Mercaptotetrazol-1-yl)naphthalen-1-yloxy]butyryl-Arg₃-NH₂Benzotriazol-5-ylcarbonyl-4-Abz-Gly₂-Arg₆-NH₂ (SEQ ID NO: 60)Benzotriazol-5-ylcarbonyl-4-Abz-Gly₂-Arg₄-NH₂ (SEQ ID NO: 61)Benzotriazol-5-ylcarbonyl-4-Abz-Gly₂-Arg₃-NH₂ (SEQ ID NO: 62)4-[5-Bromo-6-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxymethyl]benzoyl-Arg₃-NH₂4-[5-Bromo-6-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxymethyl]benzoyl-Arg₄-NH₂ (SEQ ID NO: 27)4-[5-Bromo-6-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxymethyl]benzoyl-Arg₅-NH₂ (SEQ ID NO: 26)3′,5′-Dichloro-4′-(2,4-dioxothiazolidin-5-ylidenemethyl)biphenyl-4-oyl-Arg₃-NH₂3′,5′-Dichloro-4′-(2,4-dioxothiazolidin-5-ylidenemethyl)biphenyl-4-oyl-Arg₄-NH₂(SEQ ID NO: 27)3′,5′-Dichloro-4′-(2,4-dioxothiazolidin-5-ylidenemethyl)biphenyl-4-oyl-Arg₅-NH₂(SEQ ID NO: 26)2-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)acetyl-Arg₃-NH₂2-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)acetyl-Arg₄-NH₂ (SEQ ID NO:27) 2-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)acetyl-Arg₅-NH₂ (SEQ IDNO: 26) 4-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)butyryl-Arg₅-NH₂(SEQ ID NO: 26)4-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)butyryl-Arg₄-NH₂ (SEQ IDNO: 27) 4-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)butyryl-Arg₃-NH₂5-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)pentanoyl-Arg₅-NH₂ (SEQ IDNO: 26) 5-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)pentanoyl-Arg₄-NH₂(SEQ ID NO: 27)5-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)pentanoyl-Arg₃-NH₂8-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)octanoyl-Arg₅-NH₂ (SEQ IDNO: 26) 8-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)octanoyl-Arg₄-NH₂(SEQ ID NO: 27)8-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)octanoyl-Arg₃-NH₂4-[4-(5-Cyano-1H-[1,2,3]triazol-4-yl)benzoylamino]-benzoyl-Arg₆-NH₂ (SEQID NO: 59)4-[4-(5-Cyano-1H-[1,2,3]triazol-4-yl)benzoylamino]-benzoyl-Arg₅-NH₂ (SEQID NO: 26)4-[4-(5-Cyano-1H-[1,2,3]triazol-4-yl)benzoylamino]-benzoyl-Arg₄-NH₂ (SEQID NO: 27)4-[4-(5-Cyano-1H-[1,2,3]triazol-4-yl)benzoylamino]-benzoyl-Arg₃-NH₂N-[4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenyl]terephthalamoyl-Arg₅-NH₂(SEQ ID NO: 26)N-[4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenyl]terephthalamoyl-Arg₄-NH₂(SEQ ID NO: 27)N-[4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenyl]terephthalamoyl-Arg₃-NH₂

Example 663

Equilibrium Solubility. For pH-solubility profiles, a 0.6 mM humaninsulin stock solution containing 0.2 mM Zn²⁺, 30 mM phenol, 0.2 Mmannitol, 2 mM phosphate and Zn²⁺-binding ligand as required wereprepared and the pH was adjusted to the desired value corresponding tothe alkaline endpoint of the pH-solubility profile. From these stocksolutions samples were withdrawn, the pH adjusted to the desired valuein the pH 3-8 range, and samples were incubated at 23 C for 24 hours.After centrifugation (20,000 g in 20 min at 23 C) of each sample, pH wasmeasured and the solubility was determined by quantitation of insulincontents in the supernatant by SEC HPLC analysis

The effect of various concentration of the ligand BTG₂R₅ (SEQ ID NO: 1)on the pH-dependence of insulin solubility is illustrated in FIG. 1.

Example 664

The effect of increasing concentrations of the ligand BTG₂R₄ (SEQ ID NO:2) on the pH-dependence of insulin solubility is illustrated in FIG. 2.The solubility was determined as in example 24. Solution conditions: 0.6mM human insulin, 0.2 mM mM Zn²⁺, 30 mM phenol, 0.2 M mannitol, 2 mMphosphate, 23 C.

Example 665

The slow release (prolonged action) properties of certain formulationsof the present invention was characterized by the disappearance ratefrom the subcutaneous depot following subcutaneous injections in pigs.T_(50%) is the time when 50% of the A14 Tyr(¹²⁵I) insulin hasdisappeared from the site of injection as measured with an externalγ-counter (Ribel et al., The Pig as a Model for Subcutaneous Absorptionin Man. In: M. Serrano-Rtios and P. J. Lefebre (Eds): Diabetes (1985)Proceedings of the 12^(th) congress of the International DiabetesFederation, Madrid, Spain, 1985 (Excerpta Medica, Amsterdam (1986),891-896). The composition of a series of protracted formulations isgiven in the table below together with the T_(50%) values. Thedisappearance curves are illustrated in FIG. 3 a-d. For comparison, theT_(50%) for the corresponding insulin preparations formulated withoutthe ligands would be about 2 hours.

The induction of slow release by addition of exogenous ligands of theinvention affords further advantages in terms of versatility regardingthe choice of insulin species and release patterns. Consequently, humanor mutant insulins such as Asp^(B28), Lys^(B28)Pro^(B29), orGly^(A21)Lys^(B3)Glu^(B29) may be formulated as slow- or dual-releasepreparations by adding variable amounts of His^(B10)Zn²⁺-site ligand.This is illustrated below for Asp^(B28) human insulin employing twodifferent levels of the ligand TZD-Abz-G₂R₅ (SEQ ID NO: 4) (example647). As shown in the table and in FIG. 3 panels e-f, addition of thisligand in slight excess of the Zn²⁺ concentration produces a slowrelease preparation with T_(50%) about 14.8. In contrast, when theligand is added in concentrations lower than that of Zn²⁺, a distinctlydual-release formulation results.

¹²⁵I-Prep. 1 ¹²⁵I-Prep. 2 ¹²⁵I-Prep. 3 ¹²⁵I-Prep. 4 ¹²⁵I-Prep. 5¹²⁵I-Prep. 6 Insulin (mM) 0.6 human 0.6 human 0.6 human 0.6 human 0.6Asp^(B28) 0.6 Asp^(B28) insulin insulin insulin insulin insulin insulinZn²⁺ (mM) 0.3 0.3 0.3 0.3 0.3 0.3 Phenolic 30 mM 30 mM 30 mM 30 mM 7- 30mM 30 mM ligand phenol phenol phenol hydroxyindole phenol phenol Zn²⁺ligand 6 mM BTG₂R₄ 6 mM BTG₂R₆ 2 mM BT- 2 mM BT- 0.4 mM TZD- 0.15 mMTZD- (Ex. 641) (Ex. 643) AbzG₂R₅ AbzG₂R₅ AbzG₂R₅ AbzG₂R₅ (SEQ ID (SEQ ID(Ex. 644) (Ex. 644) (Ex. 647) (Ex. 647) NO: 2) NO: 5) (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 3) NO: 3) NO: 4) NO: 4) Mannitol (mM) 112 112 150150 154 176 Phosphate 2 2 2 2 2 2 buffer (mM) pH 7.4 7.4 7.4 7.4 7.4 7.4T_(50%) (hrs) 10.2 10.3 >22 20.2 14.8 biphasic

ANALYTICAL METHODS

Assays to quantify the binding affinity of ligands to the metal site ofthe insulin R₆ hexamers:

4H3N-Assay:

The binding affinity of ligands to the metal site of insulin R₆ hexamersare measured in a UV/vis based displacement assay. The UV/vis spectrumof 3-hydroxy-4-nitro benzoic acid (4H3N) which is a known ligand for themetal site of insulin R₆ shows a shift in absorption maximum upondisplacement from the metal site to the solution (Huang et al., 1997,Bio-chemistry 36, 9878-9888). Titration of a ligand to a solution ofinsulin R₆ hexamers with 4H3N mounted in the metal site allows thebinding affinity of these ligands to be determined following thereduction of absorption at 444 nm.

A stock solution with the following composition 0.2 mM human insulin,0.067 mM Zn-acetate, 40 mM phenol, 0.101 mM 4H3N is prepared in a 10 mLquantum as described below. Buffer is always 50 mM tris buffer adjustedto pH=8.0 with NaOH/ClO₄ ⁻.

1000 μL of 2.1 mM human insulin in buffer

66.7 μL of 10 mM Zn-acetate in buffer

800 μL of 500 mM phenol in H₂O

201 μL of 4H3N in H₂O

7.93 ml buffer

The ligand is dissolved in DMSO to a concentration of 20 mM.

The ligand solution is titrated to a cuvette containing 2 mL stocksolution and after each addition the UV/vis spectrum is measured. Thetitration points are listed in Table 3 below.

TABLE 3 ligand addition ligand conc. dilution (μl) (mM) factor 1 0.0101.0005 1 0.020 1.0010 1 0.030 1.0015 2 0.050 1.0025 5 0.100 1.0050 100.198 1.0100 20 0.392 1.0200 20 0.583 1.0300 20 0.769 1.0400 20 0.9521.0500

The UV/vis spectra resulting from a titration of the compound3-hydroxy-2-naphthoic acid is shown in FIG. 5. Inserted in the upperright corner is the absorbance at 444 nm vs. the concentration ofligand.

The following equation is fitted to these datapoints to determine thetwo parameters K_(D) (obs), the observed dissociation constant, andabs_(max) the absorbance at maximal ligand concentration.abs([ligand]_(free))=(abs_(max)*[ligand]_(free))/(K_(D)(obs)+[ligand]_(free))

The observed dissociation constant is recalculated to obtain theapparent dissociation constantK _(D)(app)=K _(D)(obs)/(1+[4H3N]/K _(4H3N))

The value of K_(4H3N)=50 μM is taken from Huang et al., 1997,Biochemistry 36, 9878-9888.

TZD-Assay:

The binding affinity of ligands to the metal site of insulin R6 hexamersare measured in a fluorescense based displacement assay. Thefluorescence of 5-(4-dimethylaminobenzylidene)thiazolidine-2,4-dione(TZD) which is a ligand for the metal site of insulin R₆ is quenchedupon displacement from the metal site to the solution. Titration of aligand to a stock solution of insulin R₆ hexamers with this compoundmounted in the metal site allows the binding affinity of these ligandsto be determined measuring the fluorescence at 455 nm upon excitation at410 nm.

Preparation

Stock solution: 0.02 mM human insulin, 0.007 mM Zn-acetate, 40 mMphenol, 0.01 mM TZD in 50 mM tris buffer adjusted to pH=8.0 withNaOH/ClO₄ ⁻. The ligand is dissolved in DMSO to a concentration of 5 mMand added in aliquots to the stock solution to final concentrations of0-250 mM.

Measurements

Fluorescence measurements were carried out on a Perkin ElmerSpectrofluorometer LS50B. The main absorption band was excited at 410 nmand emission was detected at 455 nm. The resolution was 10 nm and 2.5 nmfor excitation and emission, respectively.

Data Analysis

This equation is fitted to the datapointsΔF(455 nm))=ΔF _(max)*[ligand]_(free)/(K _(D)(app)*(1+[TZD]/K_(TZD))+[ligand]_(free)))K_(D)(app) is the apparent dissociation constant and F_(max) is thefluorescence at maximal ligand concentration. The value of K_(TZD) ismeasured separately to 230 nM

Two different fitting-procedures can be used. One in which bothparameters, K_(D)(app) and F_(max), are adjusted to best fit the dataand a second in which the value of F_(max) is fixed (F_(max)=1) and onlyK_(D)(app) is adjusted. The given data are from the second fittingprocedure. The Solver module of Microsoft Excel can be used to generatethe fits from the datapoints.

1. A zinc-binding ligand of the following formula (III)A-B-C-D-X  (III) wherein: A is a chemical group of the following formulawhich reversibly binds to a His^(B10) Zn²⁺ site of an insulin hexamer;

wherein A¹ is a valence bond; AR¹ is the group

where C1 is attached to the nitrogen; C¹ is CH2; AR² is phenyl; B is alinker selected from the group consisting of A valence bond and Achemical group G^(B) of the formula —B¹—B²—C(O)—, —B¹—B²—SO₂—,—B¹—B²—CH₂—, or —B¹—B²—NH—; wherein B¹ is a valence bond, —O—, —S—, or—NR⁶—, B² is a valence bond, C₁-C₁₈-alkylene, C₂-C₁₈-alkenylene,C₂-C₁₈-alkynylene, arylene, heteroarylene, —C₁-C₁₈-alkyl-aryl-,—C₂-C₁₈-alkenyl-aryl-, —C₂-C₁₈-alkynyl-aryl-,—C(═O)—C₁-C₁₈-alkyl-C(═O)—, —C(═O)—C₁-C₁₈-alkenyl-C(═O)—,—C(═O)—C₁-C₁₈-alkyl-O—C₁-C₁₈-alkyl-C(═O)—,—C(═O)—C₁-C₁₈-alkyl-S—C₁-C₁₈-alkyl-C(═O)—,—C(═O)—C₁-C₁₈-alkyl-NR⁶—C₁-C₁₈-alkyl-C(═O)—, —C(═O)-aryl-C(═O)—,—C(═O)-heteroaryl-C(═O)—; wherein the alkylene, alkenylene, andalkynylene moieties are optionally substituted with —CN, —CF₃, —OCF₃,—OR⁶, or —NR⁶R⁷ and the arylene and heteroarylene moieties areoptionally substituted with halogen, —C(O)OR⁶, —C(O)H, OCOR⁶, —SO₂, —CN,—CF₃, —OCF₃, —NO₂, —OR⁶, —NR⁶R⁷, C₁-C₁₈-alkyl, or C₁-C₁₈-alkanoyl; R⁶and R⁷ are independently H, C₁-C₄-alkyl; C is a fragment consisting of 0to 5 neutral amino acids, wherein the individual neutral amino acids arethe same or different D is a fragment consisting of 1 to 20 basic aminoacids independently selected from the group consisting of Lys and Arg orD-isomers thereof; and X is —OH, —NH₂ or a diamino group, or a saltthereof with a pharmaceutically acceptable acid or base, or any opticalisomer or mixture of optical isomers, racemic mixture, or tautomericforms thereof.
 2. A zinc-binding ligand according to claim 1 wherein A¹is a valence bond, C₁-C₆-alkylene, —NH—C(═O)-A²-, —C₁-C₆-alkyl-S—,—C₁-C₆-alkyl-O—-, or —C(═O)—, wherein any C₁-C₆-alkyl moiety isoptionally substituted by R^(1A).
 3. A zinc-binding ligand according toclaim 2 wherein A¹ is a valence bond, C₁-C₆-alkylene, —NH—C(═O)-A²-,—C₁-C₆-alkyl-S—, or —C₁-C₆-alkyl-O, wherein any C₁-C₆-alkyl moiety isoptionally substituted by R^(1A).
 4. A zinc-binding ligand according toclaim 3 wherein A¹ is a valence bond, C₁-C₆-alkylene, or —NH—C(═O)-A²,wherein any C₁-C₆-alkyl moiety is optionally substituted by R^(1A).
 5. Azinc-binding ligand according to claim 4 wherein A¹ is a valence bond orC₁-C₆-alkylene, wherein any C₁-C₆-alkyl moiety is optionally substitutedby R^(1A).
 6. A zinc-binding ligand according to claim 5 wherein A¹ is avalence bond.
 7. A zinc-binding ligand according to claim 1 wherein A²is a valence bond or —C₁-C₆-alkyl-O—.
 8. A zinc-binding ligand accordingto claim 7 wherein A² is a valence bond.
 9. A zinc-binding ligandaccording to claim 1 wherein AR¹ is arylene or heteroarylene, whereinthe aryl or heteroaryl moieties are optionally substituted by one ormore R^(1B) independently.
 10. A zinc-binding ligand according to claim9 wherein AR¹ is selected from the group of compounds consisting ofphenylene, biphenylylene, naphthylene, anthra-cenylene,phenanthrenylene, fluorenylene, indenylene, azulenylene, furylene,thienylene, pyrrolylene, oxazolylene, thiazolylene, imidazolylene,isoxazolylene, isothiazolylene, 1,2,3-triazolylene, 1,2,4-triazolylene,pyranylene, pyridylene, pyridazinylene, pyrimidinylene, pyrazinylene,1,2,3-triazinylene, 1,2,4-triazinylene, 1,3,5-triazinylene,1,2,3-oxadiazolylene, 1,2,4-oxadiazolylene, 1,2,5-oxadiazolylene,1,3,4-oxadiazolylene, 1,2,3-thiadiazolylene, 1,2,4-thiadiazolylene,1,2,5-thiadiazolylene, 1,3,4-thiadiazolylene, tetrazolylene,thiadiazinylene, indolylene, isoindolylene, benzofurylene,benzothienylene, indazolylene, benzimidazolylene, benzthiazolylene,benzisothiazolylene, benzoxazolylene, benzisoxazolylene, purinylene,quinazolinylene, quinolizinylene, quinolinylene, isoquinolinylene,quinoxalinylene, naphthyridinylene, pteridinylene, carbazolylene,azepinylene, diazepinylene, and acridinylene, optionally substituted byone or more R^(1B) independently.
 11. A zinc-binding ligand according toclaim 10 wherein AR¹ is selected from the group of compounds consistingof phenylene, biphenylylene, naphthylene, pyridinylene, fyrylene,indolylene and carbazolylene, optionally substituted by one or moreR^(1B) independently.
 12. A zinc-binding ligand according to claim 11wherein AR¹ is selected from the group of compounds consisting ofphenylene, indolylene and carbazolylene, optionally substituted by oneor more R^(1B) independently.
 13. A zinc-binding ligand according toclaim 12 wherein AR¹ is phenylene optionally substituted by one or moreR^(1B) independently.
 14. A zinc-binding ligand according to claim 12wherein AR¹ is indolylene optionally substituted by one or more R^(1B)independently.
 15. A zinc-binding ligand according to claim 14 whereinAR¹ is


16. A zinc-binding ligand according to claim 12 wherein AR¹ iscarbazolylene optionally substituted by one or more R^(1B)independently.
 17. A zinc-binding ligand according to claim 16 whereinAR¹ is


18. A zinc-binding ligand according to claim 1 wherein R^(1B) isselected from the group consisting of hydrogen, halogen, —CN, —CF₃,—OCF₃, —NO₂, —OR^(1C), —NR^(1C)R^(1D), —SR^(1C), —S(O)₂R^(1C),—NR^(1C)C(O)R^(1D), —OC₁-C₆-alkyl-C(O)NR^(1C)R^(1D),—C₂-C₆-alkenyl-C(═O)OR^(1C), —C(O)OR^(1C), ═O, —NH—C(═O)—O—C₁-C₆-alkyl,or —NH—C(═O)—C(═O)—O—C₁-C₆-alkyl C₁-C₆-alkyl or C₂-C₆-alkenyl which isoptionally substituted with one or more substituents selected from thegroup consisting of halogen, —CN, —CF₃, —OCF₃, —OR^(1C), and—NR^(1C)R^(1D) and aryl, aryloxy, aryl-C₁-C₆-alkoxy, aryl-C₁-C₆-alkyl,aryl-C₂-C₆-alkenyl, heteroaryl, heteroaryl-C₁-C₆-alkyl, orheteroaryl-C₂-C₆-alkenyl of which the cyclic moieties are optionallysubstituted with one or more substituents selected from halogen,—C(O)OR^(1C), —CN, —CF₃, —OCF₃, —NO₂, —OR^(1C), —NR^(1C)R^(1D) andC₁-C₆-alkyl.
 19. A zinc-binding ligand according to claim 18 whereinR^(1B) is selected from the group consisting of hydrogen, halogen, —CF₃,—NO₂, —OR^(1C), —NR^(1C)R^(1D), —C(O)OR^(1C), ═O,—NH—C(═O)—O—C₁-C₆-alkyl, or —NH—C(═O)—C(═O)—O—C₁-C₆-alkyl andC₁-C₆-alkyl.
 20. A zinc-binding ligand according to claim 1 whereinR^(1C) and R^(1D) independently are hydrogen, C₁-C₆-alkyl, or aryl,wherein the aryl moieties may optionally be substituted by halogen or—COOH.
 21. A zinc-binding ligand according to claim 20 wherein R^(1C)and R^(1D) independently are hydrogen, methyl, ethyl, or phenyl, whereinthe phenyl moieties may optionally be substituted by halogen or —COOH.22. A zinc-binding ligand according to claim 1 wherein C¹ is a valencebond, C₁-C₆-alkylene, —NH—C(═O)—, —C(═O)—NH—, —C(═O)—, or—C₁-C₆-alkyl-C(═O)—N(R^(1E))— wherein the alkyl moieties are optionallysubstituted by one or more R^(1F) independently.
 23. A zinc-bindingligand according to claim 22 wherein C¹ is a valence bond, —CH₂—,—CH₂—CH₂—, —CH₂—O—, —CH₂—CH₂—O—, —CH₂—NH—, —CH₂—CH₂—NH—, —NH—CH₂—,—NH—CH₂—CH₂—, —NH—C(═O)—, —C(═O)—NH—, —O—CH₂—, —O—CH₂—CH₂—, or —C(═O)—.24. A zinc-binding ligand according to claim 1 wherein R^(1E) and R^(1F)are independently selected from C₁-C₆-alkyl.
 25. A zinc-binding ligandaccording to claim 1 wherein AR² is a valence bond C₁-C₆-alkylene,wherein the alkyl is optionally substituted by one or more R^(2A)independently arylene, aryl-C₁-C₆-alkyl, heteroarylene, wherein thearylene and heteroarylene moieties are optionally substituted by one ormore R^(2A) independently.
 26. A zinc-binding ligand according to claim25 wherein AR² is a valence bond C₁-C₆-alkylene, wherein the alkyl isoptionally substituted by one or more R^(2A) independently phenyl,phenyl-C₁-C₆-alkyl, wherein the phenyl moieties are optionallysubstituted by one or more R^(2A) independently.
 27. A zinc-bindingligand according to claim 1 wherein R^(2A) is C₁-C₆-alkyl, C₁-C₆-alkoxy,aryl, aryloxy, heteroaryl, —C₁-C₆-alkyl-COOH, —O—C₁-C₆-alkyl-COOH,—S(O)₂R^(2B), —C₂-C₆-alkenyl-COOH, —OR^(2B), —NO₂, halogen, —COOH, —CF₃,—CN, —N(R^(2B)R^(2C)), wherein the aryl or heteroaryl moieties areoptionally substituted by one or more C₁-C₆-alkyl, C₁-C₆-alkoxy,—C₁-C₆-alkyl-COOH, —C₂-C₆-alkenyl-COOH, —OR^(2B), —NO₂, halogen, —COOH,—CF₃, —CN, or —N(R^(2B)R^(2C)).
 28. A zinc-binding ligand according toclaim 27 wherein R^(2A) is C₁-C₆-alkyl, C₁-C₆-alkoxy, aryl, —OR^(2B),—NO₂, halogen, —COOH, —CF₃, —CN, —N(R^(2B)R^(2C)), wherein the aryl isoptionally substituted by one or more C₁-C₆-alkyl, C₁-C₆-alkoxy,—OR^(2B), —NO₂, halogen, —COOH, —CF₃, —CN, or —N(R^(2B)R^(2C)).
 29. Azinc-binding ligand according to claim 28 wherein R^(2A) is C₁-C₆-alkyl,C₁-C₆-alkoxy, aryl, halogen, —CF₃, wherein the aryl is optionallysubstituted by one or more C₁-C₆-alkyl, halogen, —COOH, —CF₃, or —CN.30. A zinc-binding ligand according to claim 29 wherein R^(2A) isC₁-C₆-alkyl, C₁-C₆-alkoxy, phenyl, halogen, —CF₃, wherein the phenyl isoptionally substituted by one or more C₁-C₆-alkyl, halogen, —COOH, —CF₃,or —CN.
 31. A zinc-binding ligand according to claim 1, wherein G^(B) isof the formula B¹—B²—C(O)—, B¹—B²—SO₂— or —B¹—B²—CH₂—.
 32. Azinc-binding ligand according to claim 1, wherein G^(B) is of theformula B¹—B²—C(O)—, B¹—B²—SO₂— or B¹—B²—NH—.
 33. A zinc-binding ligandaccording to claim 1, wherein G^(B) is of the formula B¹—B²—C(O)—,—B¹—B²—CH₂— or B¹—B²—NH.
 34. A zinc-binding ligand according to claim31, wherein G^(B) is of the formula B¹—B²—C(O)— or B¹—B²—SO₂.
 35. Azinc-binding ligand according to claim 31, wherein G^(B) is of theformula B¹—B²—C(O)— or —B¹—B²—CH₂—.
 36. A zinc-binding ligand accordingto claim 32 wherein G^(B) is of the formula B¹—B²—C(O)— or B¹—B²—NH—.37. A zinc-binding ligand according to claim 34, wherein G^(B) is of theformula B¹—B²—C(O)—.
 38. A zinc-binding ligand according to claim 1wherein B¹ is a valence bond, —O—, or —S—.
 39. A zinc-binding ligandaccording to claim 1 wherein B¹ is a valence bond, —O—, or —N(R⁶)—. 40.A zinc-binding ligand according to claim 1 wherein B¹ is a valence bond,—S—, or —N(R⁶)—.
 41. A zinc-binding ligand according to claim 1 whereinB¹ is —O—, —S— or —N(R⁶)—.
 42. A zinc-binding ligand according to claim38 wherein B¹ is a valence bond or —O—.
 43. A zinc-binding ligandaccording to claim 38 wherein B¹ is —O— or —S—.
 44. A zinc-bindingligand according to claim 39 wherein B¹ is —O— or —N(R⁶)—.
 45. Azinc-binding ligand according to claim 42, wherein B¹ is —O—.
 46. Azinc-binding ligand according to claim 1, wherein B² is a valence bond,C₁-C₁₈-alkylene, C₂-C₁₈-alkenylene, C₂-C₁₈-alkynylene, arylene,heteroarylene, —C₁-C₁₈-alkyl-aryl-, —C(═O)—C₁-C₁₈-alkyl-C(═O)—,—C(═O)—C₁-C₁₈-alkyl-NR⁶—C₁-C₁₈-alkyl-C(═O)—; and the alkylene moietiesare optionally substituted with —CN, —CF₃, —OCF₃, —OR⁶, or —NR⁶R⁷ andthe arylene moieties are optionally substituted with halogen, —C(O)OR⁶,—C(O)H, OCOR⁶, —SO₂, —CN, —CF₃, —OCF₃, —NO₂, —OR⁶, —NR⁶R⁷, C₁-C₁₈-alkyl,or C₁-C₁₈-alkanoyl.
 47. A zinc-binding ligand according to claim 46,wherein B² is a valence bond, C₁-C₁₈-alkylene, C₂-C₁₈-alkenylene,C₂-C₁₈-alkynylene, arylene, heteroarylene, —C₁-C₁₈-alkyl-aryl-,—C(═O)—C₁-C₁₈-alkyl-C(═O)—, —C(═O)—C₁-C₁₈-alkyl-O—C₁-C₁₈-alkyl-C(═O)—,and the alkylene moieties are optionally substituted with —CN, —CF₃,—OCF₃, —OR⁶, or —NR⁶R⁷ and the arylene moieties are optionallysubstituted with halogen, —C(O)OR⁶, —C(O)H, OCOR⁶, —SO², —CN, —CF₃,—OCF₃, —NO₂, —OR⁶, —NR⁶R⁷, C₁-C₁₈-alkyl, or C₁-C₁₈-alkanoyl.
 48. Azinc-binding ligand according to claim 47, wherein B² is a valence bond,C₁-C₁₈-alkylene, C₂-C₁₈-alkenylene, C₂-C₁₈-alkynylene, arylene,heteroarylene, —C₁-C₁₈-alkyl-aryl-, —C(═O)—C₁-C₁₈-alkyl-C(═O)—, and thealkylene moieties are optionally substituted with —CN, —CF₃, —OCF₃,—OR⁶, or —NR⁶R⁷ and the arylene moieties are optionally substituted withhalogen, —C(O)OR⁶, —C(O)H, OCOR⁶, —SO₂, —CN, —CF₃, —OCF₃, —NO₂, —OR⁶,—NR⁶R⁷, C₁-C₁₈-alkyl, or C₁-C₁₈-alkanoyl.
 49. A zinc-binding ligandaccording to claim 48, wherein B² is a valence bond, C₁-C₁₈-alkylene,arylene, heteroarylene, —C₁-C₁₈-alkyl-aryl-, —C(═O)—C₁-C₁₈-alkyl-C(═O)—,and the alkylene moieties are optionally substituted with —CN, —CF₃,—OCF₃, —OR⁶, or —NR⁶R⁷ and the arylene moieties are optionallysubstituted with halogen, —C(O)OR⁶, —C(O)H, OCOR⁶, —SO₂, —CN, —CF₃,—OCF₃, —NO₂, —OR⁶, —NR⁶R⁷, C₁-C₁₈-alkyl, or C₁-C₁₈-alkanoyl.
 50. Azinc-binding ligand according to claim 49 wherein B² is a valence bond,C₁-C₁₈-alkylene, arylene, heteroarylene, —C₁-C₁₈-alkyl-aryl-, and thealkylene moieties are optionally substituted with —CN, —CF₃, —OCF₃,—OR⁶, or —NR⁶R⁷ and the arylene moieties are optionally substituted withhalogen, —C(O)OR⁶, —C(O)H, OCOR⁶, —SO₂, —CN, —CF₃, —OCF₃, —NO₂, —OR⁶,—NR⁶R⁷, C₁-C₁₈-alkyl, or C₁-C₁₈-alkanoyl.
 51. A zinc-binding ligandaccording to claim 50, wherein B² is a valence bond, C₁-C₁₈-alkylene,arylene, —C₁-C₁₈-alkyl-aryl-, and the alkylene moieties are optionallysubstituted with —CN, —CF₃, —OCF₃, —OR⁶, or —NR⁶R⁷ and the arylenemoieties are optionally substituted with halogen, —C(O)OR⁶, —C(O)H,OCOR⁶, —SO₂, —CN, —CF₃, —OCF₃, —NO₂, —OR⁶, —NR⁶R⁷, C₁-C₁₈-alkyl, orC₁-C₁₈-alkanoyl.
 52. A zinc-binding ligand according to claim 51,wherein B² is a valence bond or —C₁-C₁₈-alkylene, and the alkylenemoieties are optionally substituted moieties are optionally substitutedwith —CN, —CF₃, —OCF₃, —OR⁶, or —NR⁶R⁷ and the arylene moieties areoptionally substituted with halogen, —C(O)OR⁶, —C(O)H, OCOR⁶, —SO₂, —CN,—CF₃, —OCF₃, —NO₂, —OR⁶, —NR⁶R⁷, C₁-C₁₈-alkyl, or C₁-C₁₈-alkanoyl.
 53. Azinc-binding ligand according to claim 1, wherein C consists of 0 to 5neutral amino acids independently selected from the group consisting ofAbz, Gly, Ala, Thr, and Ser.
 54. A zinc-binding ligand according toclaim 53, wherein C consists of 0 to 5 Gly.
 55. A zinc-binding ligandaccording to claim 54, wherein C consists of 1 Gly.
 56. A zinc-bindingligand according to claim 54, wherein C consists of 2 Gly.
 57. Azinc-binding ligand according to claim 54, wherein C consists of 3 Gly.58. A zinc-binding ligand according to claim 54 wherein C consists of 4Gly.
 59. A zinc-binding ligand according to claim 54, wherein C consistsof 5 Gly.
 60. A zinc-binding ligand according to claim 1, wherein thepositively charged groups of D number from 1 to
 16. 61. A zinc-bindingligand according to claim 60, wherein the positively charged groups of Dnumber from 1 to
 12. 62. A zinc-binding ligand according to claim 61,wherein the positively charged groups of D number from 1 to
 10. 63. Azinc-binding ligand according to claim 1, wherein the basic amino acidis Arg.
 64. A zinc-binding ligand according to claim 1, wherein X is —OHor —NH₂.
 65. A zinc-binding ligand according to claim 64, wherein X is—NH₂.
 66. Method of prolonging the action of an insulin preparationwhich comprises adding a zinc-binding ligand according to claim 1 to theinsulin preparation.
 67. A method of preparing a zinc-binding ligandaccording to claim 1 comprising the steps of Identifying startercompounds that are able to displace a ligand from the R-stateHis^(B10)-Zn²⁺ site optionally attaching a fragment consisting of 0 to 5neutral α- or β-amino acids attaching a fragment comprising 1 to 20positively charged groups independently selected from amino or guanidinogroups.